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Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear...

Arteriovenous (AV) fistulas for hemodialysis can lead to cardiac volume loading and increased serum brain natriuretic peptide (BNP) levels. Whether short-term AV loop placement in patients undergoing microsurgery has an impact on cardiac biomarkers and circulating microRNAs (miRNAs), potentially indicating an increased hemodynamic risk, remains elusive. Fifteen patients underwent AV loop placement with delayed free flap anastomosis for microsurgical reconstructions of lower extremity soft-tissue defects. N-terminal pro-BNP (NT-proBNP), copeptin (CT-proAVP), and miRNA expression profiles were determined in the peripheral blood before and after AV loop placement. MiRNA expression in the blood was correlated with miRNA expression from AV loop vascular tissue. Serum NT-proBNP and copeptin levels exceeded the upper reference limit after AV loop placement, with an especially strong NT-proBNP increase in patients with preexistent cardiac diseases. A miRNA signature of 4 up-regulated (miR-3198, miR-3127-5p, miR-1305, miR-1288-3p) and 2 down-regulated miRNAs (miR30a-5p, miR-145-5p) which are related to cardiovascular physiology, showed a significant systemic deregulation in blood and venous tissue after AV loop placement. AV loop placement causes serum elevations of NT-proBNP, copeptin as well as specific circulating miRNAs, indicating a potentially increased hemodynamic risk for patients with cardiovascular comorbidities, if free flap anastomosis is delayed.

Background MicroRNAs (miRNAs) are small RNAs regulating gene expression post-transcriptionally. While acquired changes of miRNA and mRNA profiles in cancer have been extensively studied, little is known about expression changes of circulating miRNAs and messenger RNAs (mRNA) in monogenic constitutional anomalies affecting several organ systems, like Marfan syndrome (MFS). We performed integrated miRNA and mRNA expression profiling in blood samples of Marfan patients in order to investigate deregulated miRNA and mRNA networks in these patients which could serve as potential diagnostic and prognostic tools for MFS therapy. Methods MiRNA and mRNA expression profiles were determined in blood samples from MFS patients (n = 7) and from healthy volunteer controls (n = 7) by microarray analysis. Enrichment analyses of altered mRNA expression were identified using bioinformatic tools. Results A total of 28 miRNAs and 32 mRNAs were found to be significantly altered in MFS patients compared to controls (> 2.0-fold change, adjusted P

Description, Vitamin D–activated Trem2+ macrophages provide a novel strategy to develop advanced cell therapies for complex wounds., Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available. The relationship between the immune response to foreign grafts and their impact on wound healing has not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSGs) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to a fibrotic collagen deposition. We show that macrophage differentiation in response to hSTSG implantation is driven toward regenerative Trem2+ subpopulations and found that hydrogel delivery of these cells significantly accelerated wound closure. Our study identifies the preclinical therapeutic potential of Trem2+ macrophages to mitigate fibrosis and promote wound healing, providing a novel effective strategy to develop advanced cell therapies for complex wounds.

BACKGROUND Reinforcement of the abdominal wall with synthetic mesh in autologous breast reconstruction using abdominal free tissue transfer decreases the risk of bulging and herniation. However, the impact of the plane of mesh placement on donor site complications has not yet been investigated. METHODS We performed a retrospective analysis of 312 patients who had undergone autologous breast reconstruction with muscle-sparing transverse rectus abdominis myocutaneous (MS-TRAM) flaps or deep inferior epigastric perforator (DIEP) flaps as well as polypropylene mesh implantation at the donor site. Donor site complications were compared among patients with different flap types and different mesh positions including overlay (n = 90), inlay and overlay (I-O; n = 134), and sublay (n = 88). RESULTS Abdominal hernias occurred in 2.86% of patients who had undergone MS-TRAM reconstructions and in 2.63% of patients who had undergone DIEP reconstructions. When comparing patients with different mesh positions, donor site complications occurred in 14.4% of patients with overlay mesh, 13.4% of patients with I-O mesh, and 10.2% of patients with sublay mesh (P = 0.68). Abdominal hernias occurred in 4.44% of patients with overlay mesh, 2.24% of patients with I-O mesh, and 2.27% of patients with sublay mesh (P = 0.69). Multivariable logistic regression analysis did not identify a significant association between mesh position and hernia rates as well as wound complications. CONCLUSIONS Our data indicate that the plane of synthetic mesh placement in relation to the rectus abdominis muscle does not impact the rate of postoperative donor site complications in patients undergoing breast reconstruction with MS-TRAM or DIEP flaps.

Background In elderly patients, complex soft tissue defects are increasingly observed due to the prolonged life expectancy and accompanying comorbidities. The aim of this study is to evaluate whether free tissue transfer is safe in very old patients without additional risk and complications. Methods All patients older than 65 years undergoing free tissue transfer between November 2007 and September 2016 were reviewed in a retrospective study. Two cohorts were compared regarding perioperative morbidity and postoperative outcome (cohort 1 [old patients, ages 65–79]; cohort 2 [very old patients, ages ≥ 80]). Results In total, 256 patients were included in the study (cohort 1 [n = 217]; cohort 2 [n = 39]). Overall, 262 free flaps were performed due to a second microsurgical reconstruction in six cases. No statistically significant differences between cohorts were observed regarding surgical complications, total flap losses, and mortality. Detailed evaluation of cohort 2 revealed a significant learning curve during the observation period regarding the perioperative management and procedure of soft tissue reconstruction: operation length as well as postoperative intensive care unit stay decreased significantly over time (p Conclusion Our study showed that age is not associated with an increased risk of postoperative complications. Reliable muscle free flaps, two-stage procedures, and safe vascular supply are important strategic aspects to achieve microvascular tissue transfer with high success rates in geriatric patients.

Quality of life (QOL) is an important outcome measure after ventral hernia repair (VHR). The Hernia-Related QOL Survey (HerQLes) is a specific survey tool for QOL after VHR. Studies comparing QOL in patients with biologic mesh repairs (BMRs) and synthetic mesh repairs (SMRs) are lacking.A survey based on the HerQLes was administered via e-mail to 974 patients who had undergone VHR at Stanford Medical Center. From 175 patients who were included in the study, the mean HerQLes scores and postoperative complications were compared between patients with suture repairs (SR), BMR, SMR, with and without component separation, and different types of SMR.Quality of life was lower in patients with hernias of 50 cm or greater, obesity, history of tobacco use, previous abdominal surgeries, hernia recurrences, and postoperative complications (P0.05). Patients with SR and SMR had a comparable QOL (71.58 vs 70.12, P = 0.75). In patients with Modified Ventral Hernia Working Group grade 2 hernias, a significantly lower QOL was found after BMR compared with SMR. Postoperative complications did not significantly differ between the groups. Recurrence rates were comparable between MR (10.4%) and SR (8.3%, P = 0.79), but higher in BMR (21.7%) compared with SMR (6.6%, P0.05).Previous abdominal surgeries, previous hernia repairs, tobacco use, and hernia sizes of 50 cm or greater negatively affect QOL after VHR. Our data indicate that QOL is comparable between patients with SMR and SR, however, is lower in patients with Modified Ventral Hernia Working Group grade 2 and BMR compared with SMR, raising the benefit of BMR in light of its higher cost into question.

Purpose: The longevity of biomedical implants is limited by adverse implant-tissue interactions leading to implant failure. In almost all cases, implant failure occurs due to a phenomenon known as the foreign body response (FBR). FBR is characterized by fibrous capsule formation around implanted devices, leading to implant malfunction as well as distortion of the surrounding tissue. In the context of breast implants, FBR leads to capsular contracture, which is the cause of a significant proportion of all breast implant failures. Despite the high prevalence of FBR-mediated implant failure, the underlying mechanisms of FBR are incompletely understood. Our work utilizing mass spectrometry has demonstrated that when compared to healthy subcutaneous tissue, foreign body capsules in mice and humans display an up-regulation of IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), a scaffolding protein involved in multiple mechanotransduction pathways. Based on these findings, we sought to investigate which cellular subpopulations express IQGAP1 as well as its role in mechanotransduction pathways mediating the development of FBR. Methods: To verify the importance of IQGAP1-mediated signaling in FBR, we employed a murine model of mechanically stimulated silicone implants (MSI), which were implanted subcutaneously in wildtype (WT) and IQGAP1+/- haplo-insufficient mice to compare the effect of human levels of mechanical stress on FBR. Homozygous IQGAP1 KO mice have been previously reported to harbor a fragile phenotype, more prone to pulmonary vascular damage and gastric pathologies. Haplo-insufficient mice were utilized in these experiments in order to prevent potential systemic complications from elevated levels of mechanical stimulation produced by the MSI model. We explanted the foreign body capsules from IQGAP1+/- haplo-insufficient and WT mice and performed single-cell RNA sequencing (sc-RNA-seq) on cells isolated from the capsules. We histologically assessed the quantity as well as maturity of collagen deposition using Masson’s Trichrome and Herovici staining of tissue sections from explanted foreign body capsules. Moreover, immunostaining for the mechanotransduction related proteins alpha-smooth muscle actin (α-SMA), phosphorylated focal adhesion kinase (p-FAK), phosphorylated cell division control protein 42 (p-cdc42), and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was performed to assess the impact of IQGAP1-deficiency on the protein level. Results: We found that IQGAP1-deficient mice displayed a significantly reduced FBR as evidenced by thinner capsules, lower levels of collagen deposition, collagen maturity, and myofibroblast activation. Our scRNA-seq analysis revealed a depletion of mechanoresponsive myeloid cells in IQGAP1-deficient mice. This was confirmed on the protein level by a significantly reduced expression of a-SMA, p-FAK, p-cdc42, and p-ERK1/2 in foreign body capsule tissue from IQGAP1-deficient mice compared to WT mice. Conclusion: Our results highlight the important role of IQGAP1 as a critical early stage mediator of mechanotransduction pathways contributing to the development of FBR. Further, we show that IQGAP1 plays a role in modulating the innate immune response to synthetic implants. Therefore, IQGAP1 may be a promising target for the development of novel therapeutics to limit the development of FBR around biomedical implants.

Purpose: Dendritic cells (DCs) are a heterogeneous cell population which critically regulates the adaptive immune response. Depending on their activation status, DCs can also promote peripheral immune tolerance, thus limiting the activation of the immune system and tissue damage. The N-myc downregulated gene 2 (Ndrg2) is highly expressed in DCs and limits the secretion of vascular endothelial growth factor (VEGF), which is critical for wound healing. Cell based therapy approaches using DCs have been approved by the FDA and clinical trials using DC immunotherapy are being performed against a variety of cancer types. However, the role of DC therapy for wound healing has not yet been investigated. Methods: Hematopoietic progenitor cells were isolated from the bone marrow of mice and differentiated into DCs over a 7-day in vitro culture period. Pharmacologic down-regulation of Ndrg2 was performed by treatment of cultures with 1,25-dihydroxyvitamin D3 (VD3) and the angiogenic potential of the treated cells was evaluated by endothelial cell (EC) tube formation assays. Cytokine secretion of DCs was measured in the conditioned media using Luminex multiplex assays. To permanently knock out Ndrg2 in DCs, a CRISPR/Cas9 gene editing approach was developed, using Cas9/sgRNA-ribonucleoproteins and electroporation. The determine the impact of genetically edited DCs on wound healing, splinted excisional wounds in C57BL6/J (wild-type) mice were treated weekly with pullulan-collagen hydrogels seeded with Ndrg2-knockout (KO) DCs, control DCs which had undergone electroporation only, or blank hydrogels. The transcriptomic impact of Ndrg2 downregulation on DC fate was evaluated by microfluidic single-cell RNA sequencing (scRNA seq) of Ndrg2-KO DCs, VD3-treated DCs and control DCs. Results: Ndrg2 down-regulation lead to a significantly stronger EC tube formation in co-cultures with VD3-treated DCs, and strongly enhanced VEGF secretion compared to untreated DCs in vitro. A CRISPR/Cas9 editing pipeline was developed for KO of Ndrg2 in DCs with a transfection rate and editing efficiency of > 90% shown by Sanger Sequencing. Excisional wounds treated with Ndrg2-KO DCs demonstrated significantly accelerated healing compared to control DCs and blank hydrogels. scRNA seq revealed that Ndrg2 downregulation strongly induced Vegfa expression and anti-oxidant transcriptomic signatures. Conclusion: Our data indicate that KO of Ndrg2 in DCs strongly enhances their secretion of VEGF, thus promoting angiogenesis and accelerating wound healing. Given the ready availability of DCs from the human blood through established leukapheresis protocols and easy multiplication in vitro, CRISPR/Cas9 editing of DCs is a promising new approach to induce wound healing and soft-tissue regeneration.

Cutaneous wounds are a growing global health burden as a result of an aging population coupled with increasing incidence of diabetes, obesity, and cancer. Cell-based approaches have been used to treat wounds due to their secretory, immunomodulatory, and regenerative effects, and recent studies have highlighted that delivery of stem cells may provide the most benefits. Delivering these cells to wounds with direct injection has been associated with low viability, transient retention, and overall poor efficacy. The use of bioactive scaffolds provides a promising method to improve cell therapy delivery. Specifically, hydrogels provide a physiologic microenvironment for transplanted cells, including mechanical support and protection from native immune cells, and cell–hydrogel interactions may be tailored based on specific tissue properties. In this review, we describe the current and future directions of various cell therapies and usage of hydrogels to deliver these cells for wound healing applications.

Burn scars and scar contractures cause significant morbidity for patients. Recently, cell-based therapies have been proposed as an option for improving healing and reducing scarring after burn injury, through their known proangiogenic and immunomodulatory paracrine effects. Our laboratory has developed a pullulan-collagen hydrogel that, when seeded with mesenchymal stem cells (MSCs), improves cell viability and augments their proangiogenic capacity

Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available and have been shown to significantly accelerate wound healing. Although the cellular elements of foreign grafts are rejected, the extracellular matrix components integrate into the wound and may underlie their beneficial effects on wound healing. The molecular mechanisms defining the relationship between the immune response to foreign grafts and their impact on wound healing have not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of clinically available human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSG) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to highly aligned collagen deposition, characteristic of fibrosis and scar. Using single-cell RNA-sequencing, we show that macrophage differentiation in response to hSTSG is driven by vitamin D (VD) signaling toward Trem2+ subpopulations with an enrichment of pro-angiogenic and anti-fibrotic transcriptomic programs. We subsequently induced this regenerative subpopulation in vitro by treating bone marrow-derived cells with vitamin D3 and found that hydrogel delivery of Trem2+ macrophages significantly accelerated wound closure in a human-like murine excisional wound model. Our study identifies the preclinical therapeutic potential of Trem2+ macrophages to mitigate fibrosis and promote wound healing and provides a novel effective strategy to develop advanced cell therapies for complex wounds.One Sentence SummaryVitamin D-activated Trem2+ macrophages promote angiogenesis and mitigate fibrosis, providing a novel effective strategy to develop advanced cell therapies for complex wounds.

Burn injury in the craniofacial region causes significant health and psychosocial consequences and presents unique reconstructive challenges. Healing of severely burned skin and underlying soft tissue is a dynamic process involving many pathophysiological factors, often leading to devastating outcomes such as the formation of hypertrophic scars and debilitating contractures. There are limited treatment options currently used for post-burn scar mitigation but recent advances in our knowledge of the cellular and molecular wound and scar pathophysiology have allowed for development of new treatment concepts. Clinical effectiveness of these experimental therapies is currently being evaluated. In this review, we discuss current topical therapies for craniofacial burn injuries and emerging new therapeutic concepts that are highly translational.

Cryopreserved human skin allografts (CHSAs) are used for the coverage of major burns when donor sites for autografts are insufficiently available and have clinically shown beneficial effects on chronic non-healing wounds. However, the biologic mechanisms behind the regenerative properties of CHSA remain elusive. Furthermore, the impact of cryopreservation on the immunogenicity of CHSA has not been thoroughly investigated and raised concerns with regard to their clinical application. To investigate the importance and fate of living cells, we compared cryopreserved CHSA with human acellular dermal matrix (ADM) grafts in which living cells had been removed by chemical processing. Both grafts were subcutaneously implanted into C57BL/6 mice and explanted after 1, 3, 7, and 28 days (n = 5 per group). A sham surgery where no graft was implanted served as a control. Transmission electron microscopy (TEM) and flow cytometry were used to characterise the ultrastructure and cells within CHSA before implantation. Immunofluorescent staining of tissue sections was used to determine the immune reaction against the implanted grafts, the rate of apoptotic cells, and vascularisation as well as collagen content of the overlaying murine dermis. Digital quantification of collagen fibre alignment on tissue sections was used to quantify the degree of fibrosis within the murine dermis. A substantial population of live human cells with intact organelles was identified in CHSA prior to implantation. Subcutaneous pockets with implanted xenografts or ADMs healed without clinically apparent rejection and with a similar cellular immune response. CHSA implantation largely preserved the cellularity of the overlying murine dermis, whereas ADM was associated with a significantly higher rate of cellular apoptosis, identified by cleaved caspase-3 staining, and a stronger dendritic cell infiltration of the murine dermis. CHSA was found to induce a local angiogenic response, leading to significantly more vascularisation of the murine dermis compared with ADM and sham surgery on day 7. By day 28, aggregate collagen-1 content within the murine dermis was greater following CHSA implantation compared with ADM. Collagen fibre alignment of the murine dermis, correlating with the degree of fibrosis, was significantly greater in the ADM group, whereas CHSA maintained the characteristic basket weave pattern of the native murine dermis. Our data indicate that CHSAs promote angiogenesis and collagen-1 production without eliciting a significant fibrotic response in a xenograft model. These findings may provide insight into the beneficial effects clinically observed after treatment of chronic wounds and burns with CHSA.

Purpose: Scarring in the mouse dorsal dermis is mediated by pro-fibrotic, Engrailed-1 lineage-positive fibroblasts (EPFs). We recently showed that mechanotransduction blockade (YAP inhibition, using the drug verteporfin), results in complete wound regeneration, with full recovery of normal dermal appendages (hair follicles, glands), extracellular matrix (ECM) architecture, and tensile strength. This regenerative outcome following verteporfin treatment is mediated by Engrailed-1 lineage-negative fibroblasts (ENFs). The complex milieu of cell types and molecular signals involved in wound repair makes it difficult to study using any single data modality. Thus, we sought to use a holistic approach, incorporating multiple high-throughput, high-dimensional analyses, to define the divergent molecular events distinguishing typical scarring healing from verteporfin-induced wound regeneration. Methods: C57BL/6J mice underwent dorsal splinted excisional wounding per standard protocol. Wounds were treated with local injection of either verteporfin or vehicle control (PBS) on POD 0. We harvested unwounded skin and wounds at POD 2, 7, 14, and 30 (n=5 mice/timepoint and treatment) and subjected wound cells to three analyses: single-cell RNA-sequencing (scRNA-seq, using 10X Genomics Chromium); timsTOF, a recently-developed, high-throughput proteomic sequencing platform; and a novel machine learning algorithm for quantitatively comparing ECM ultrastructure. Results: Pseudotime analysis (Monocle3) of pooled scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (En-1 lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (En-1 lineage-negative; Rspo1, Dkk2/3, Trps1). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and fibrotic/scar ECM features. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were associated with a “basket-weave” ECM pattern quantitatively indistinguishable from that of unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed in situ hybridization (RNAScope) for Rspo1 (Wnt agonist), Trps1 (master hair follicle regulator), Ank1 (YAP target gene), and Dpp4 (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low Dpp4) in YAP-inhibited (low Ank1) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high Rpos1, Trps1). These data suggest that YAP inhibition unlocks wound regeneration via Wnt-active, En-1 lineage-negative fibroblasts. Conclusion: By studying regenerating (verteporfin-treated) versus scarring wounds across multiple healing timepoints and high-dimensional data modalities, we were able to profile fibrotic versus regenerative healing at unprecedented depth. Our integrated analysis revealed that dermal fibroblasts in these two wound settings exhibit distinct molecular trajectories defined by divergent transcriptomic, proteomic, and ultrastructural properties. Further, we found that wound regeneration in the context of verteporfin treatment is associated with suppression of mechanical signaling and activation of key Wnt pathway members including Trps1 (a gene with known hair follicle developmental roles). These results could have important implications for both the fundamental study of wound healing and potential anti-scarring therapeutic avenues

Purpose: Fractionated fat (FF) has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft tissue defects is limited due to volume loss over time. We have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel (NHC) enriched with FF. Methods: FF was generated by emulsification of groin fat pads from rats and mixed in a 3:1 ratio with NHC (NHC-FF). NHC-FF or NHC alone were placed into isolation chambers together with arteriovenous (AV) loops, which were subcutaneously implanted into the groin of rats (n=8 per group). After 21 days, animals were euthanized, systemically perfused with ink, and tissue was explanted for histological analysis. Immunofluorescent staining (IF) and confocal laser scanning microscopy were used to quantify CD34+ progenitor cells and macrophage subpopulations. Results: NHC-FF tissue maintained its shape without shrinking and showed a significantly stronger functional neovascularization compared to NHC alone at 21 days after implantation (mean vessel count: 833.5 ± 206.1 vs. 296.5 ± 114.1, p = 0.04). Tissue remodeling and cellular infiltration were greater in NHC-FF (mean cell count: 49,707 ± 18,491 vs. 9,263 ± 3,790, p = 0.005) with a significantly higher amount of progenitor cells and regenerative CD163+ macrophages compared to NHC alone. Conclusion: FF-enriched NHC transforms into highly vascularized soft tissue over 21 days without signs of shrinking and promotes macrophage polarization toward regenerative phenotypes. Enrichment of injectable NHC with FF represents a promising approach for durable reconstruction of soft tissue defects.

Hand injuries often result in significant functional impairments and are rarely completely restored. The spontaneous regeneration of injured appendages, which occurs in salamanders and newts, for example, has been reported in human fingertips after distal amputation, but this type of regeneration is rare in mammals and is incompletely understood. Here, we study fingertip regeneration by amputating murine digit tips, either distally to initiate regeneration, or proximally, causing fibrosis. Using an unbiased microarray analysis, we found that digit tip regeneration is significantly associated with hair follicle differentiation, Wnt, and sonic hedgehog (SHH) signaling pathways. Viral over-expression and genetic knockouts showed the functional significance of these pathways during regeneration. Using transgenic reporter mice, we demonstrated that, while both canonical Wnt and HH signaling were limited to epidermal tissues, downstream hedgehog signaling (through Gli) occurred in mesenchymal tissues. These findings reveal a mechanism for epidermal/mesenchyme interactions, governed by canonical hedgehog signaling, during digit regeneration. Further research into these pathways could lead to improved therapeutic outcomes after hand injuries in humans.

Small recalcitrant non-unions with poor perfusion require reconstruction with vascularized bone flaps. Cases with concomitant large soft tissue defects are especially challenging, since vascularized soft tissue transfer is often indicated and distant microvascular anastomoses may be required. We introduce a sequential chimeric free flap composed of a medial femoral condyle corticoperiosteal flap anastomosed to an anterolateral thigh flow-through flap (MFC-ALT flap) and report its use for reconstruction of small non-unions with concomitant large soft tissue defects in three exemplary patients. Two female and one male patients ages 39-58 years suffered from composite bone and soft tissue defects of the lower extremity and clavicle caused by tumor resection and postoperative radiation resp. infected tibial pilon fracture. The sizes of the soft tissue defects ranged from 15-23 × 4.5-6 cm and the sizes of the bone defects ranged from 1.5-4 × 2-4 cm. Defect reconstructions were performed in all cases with sequential chimeric MFC-ALT flaps with sizes ranging from 2-4 × 1.6-4 cm for the MFC and 21-23 × 7-8 cm for the ALT skin paddles. Functional reconstructions were achieved in all cases resulting in stable unions and soft tissue coverage enabling the patients to bear full weight without assistance on 5-months follow-up. Postoperative course was uneventful and complications were restricted to a small skin necrosis at the suture line in one case. MFC-ALT flaps may be a safe, and effective procedure for one-stage reconstructions of small, irregularly shaped bone defects with concomitant large soft tissue loss or surrounding instable scarring, particularly in cases of recalcitrant non-unions after radiation exposure.

Cutaneous scar formation following injury is an exuberant fibro-proliferative event and causes aesthetic distress associated with functional problems for those afflicted. Traditionally, surgical revision of excessive hypertrophic scars is commonly performed. However, scar recurrence rate is high and surgical treatments are often combined with radiation therapy, corticosteroid injections, or with other noninvasive therapies. Pharmacological intervention of scar-promoting cellular activities has recently gained traction based on the emerging scientific evidence proving efficacy and safety of new therapeutics in preclinical studies. Ideal new drug therapies would be noninvasive, inexpensive, safe-to-use, and effective in prevention of scar formation. Recently, cytokine-based therapies and modulators of mechanotransduction are being evaluated for their anti-fibrotic properties in the reduction and prevention of scarring. Therapeutic potential of evidence-based new scar therapeutics holds promise toward improving the current wound and scar management practice.

Shared decision-making is a key component of patient-centered care and has been shown to improve patient satisfaction and quality of life. Herein, we study the impact of a standardized patient education class for prospective breast reconstruction patients on clinic efficiency, access to care, and perception on shared decision-making.The number of new patient consultations per clinic, as well as average duration of the first individual clinic encounter, was compared before and after the introduction of a standardized education class given by the senior author to all new breast reconstruction patients. To evaluate patients' perception of shared decision-making, the 9-item Shared Decision-Making Questionnaire (SDM-Q9) and the Satisfaction with Information scale of the BREAST-Q Reconstruction Module were electronically distributed among the patients and compared between the two groups.Introduction of the patient education class was associated with a significant reduction in the duration of new patient encounters compared to historic controls (31.8 min vs. 53.5 min, p 0.01) along with permitting a 43% increase in new patient visits. No differences in self-perceived patient education and autonomy were seen between class participants and historic controls in the SDM-Q9 scores (p = 0.58) and BREAST-Q scores (p = 0.14).The introducing a standardized patient education class translated into a significant reduction in the duration of individual new patient encounters, thereby increasing patient access to care, while maintaining high-quality standards of self-perceived patient education and shared decision-making.

Background Success of free tissue transfer depends on standardized intraoperative flap design, microsurgical technique, and postoperative monitoring. We sought to investigate whether laser speckle imaging (LSI) is suitable for optimization of intraoperative flap design and postoperative monitoring of free flaps with skin paddles. Methods Skin perfusion was assessed with LSI in 27 free flaps after dissection at the donor site, after anastomosis at the recipient site, after inset and on postoperative days (POD) 1, 5, and 10. Skin perfusion of the whole flap (ROI [region of interest]-1) and the area over the pedicle (ROI-2) were compared between patients with and without postoperative complications (POC+ and POC − , respectively). A receiver operating characteristic (ROC) analysis was performed to determine the optimal cutoff for perfusion during LSI-guided microsurgery. Results In flaps without or only minor POC, intraoperatively measured perfusion over ROI-2 was significantly higher compared with ROI-1, whereas no significant differences were found for flaps with major POC. Perfusion of ROI-1 and 2 intraoperatively and on POD 1 was significantly lower in the POC+ compared with the POC− group (p 0.8 for identification of flaps with major POC. Conclusion LSI is an easy to use, noninvasive technique for identification of malperfused areas in free flaps, thus allowing for intraoperative decision-making on flap dimensions and postoperative monitoring. LSI therefore is a valuable tool for perfusion assessment with a high potential to become an established part of microsurgical practice.

BACKGROUND The optimal time for flap anastomosis to an arteriovenous loop remains controversial. Whether perforator flaps and axially vascularized muscle or fasciocutaneous flaps lead to comparable outcomes in conjunction with arteriovenous loops has not been investigated. METHODS Medical records from 103 patients undergoing arteriovenous loop reconstruction (76 one-stage and 27 two-stage) between 2007 and 2017 were reviewed. Postoperative outcomes were compared between one- and two-stage arteriovenous loop reconstructions and different types of free flaps. RESULTS Rates of flap thrombosis, major wound complications, and flap failure did not differ significantly between one- and two-stage arteriovenous loop reconstructions (14.47 percent versus 11.11 percent, p = 1.00; 30.26 percent versus 25.93 percent, p = 0.67; and 10.53 percent versus 7.41 percent, p = 1.00). For two-stage arteriovenous loop reconstructions, the time interval between arteriovenous loop placement and flap anastomosis was a predictor for thrombotic events (OR, 1.31; p < 0.05). Anterolateral thigh flaps in conjunction with arteriovenous loops showed higher failure rates (33.33 percent) compared with all other flaps (6.59 percent) (p < 0.05) and combined latissimus dorsi and parascapular flaps (0 percent) (p < 0.05). Thrombosis rates were higher in anterolateral thigh flaps (33.33 percent) compared with all other flaps (10.99 percent; p = 0.056), and combined latissimus dorsi and parascapular flaps (0 percent; p < 0.05). CONCLUSIONS Two-stage arteriovenous loop reconstructions do not lead to increased postoperative complications compared to one-stage arteriovenous loop reconstructions and may be favorable in complicated cases because of shorter operative times. To avoid an increased thrombosis risk, flap anastomosis should not be delayed beyond 10 days in two-stage arteriovenous loop reconstructions. Anterolateral thigh flaps are less suitable for arteriovenous loop reconstructions because of higher complication rates. CLINICAL QUESTION/LEVEL OF EVIDENCE Therapeutic, III.

BACKGROUND Botulinum toxin treatment for forehead wrinkles has been extensively studied and found to be a safe and reproducible procedure. However, the effect on the position of the eyebrows, which relies on the dynamic positioning of the treated muscles, has received less attention and has not been studied with 3-dimensional (3D) technology. OBJECTIVES The authors sought to evaluate the changes of eyebrow position after standardized botulinum toxin treatment of glabella or glabella and frontalis muscles with 3D imaging. METHODS In a prospective study, 2 groups of adult females were treated with botulinum toxin A at the glabella only (G) or at the glabella and the frontalis muscle (F/G). The brow position was measured at 5 positions with 3D photography before injection and 2 weeks, 3 months, and 6 months after injection. Results were statistically analyzed and related to patient age. RESULTS In the F/G group, the brow descended significantly almost across the whole brow length after 2 weeks. The descent lessened after 3 months and moved medially. No clear trend was evident in the G group. There was no significant change attributed to patient age in both groups. CONCLUSIONS Botulinum toxin treatment of the glabella and frontalis muscle impacts the position and configuration of the eyebrow. The degree of change is affected by the amount of frontalis weakening rather than by treatment of the glabella. While age in general is not a reliable predictor, individual factors play the major role in how pronounced a change of the brow can be expected. LEVEL OF EVIDENCE: 2

Background Limb salvage in patients with peripheral vascular disease (PVD) and soft tissue defects often requires both a restoration of blood flow to the lower extremity and soft tissue coverage. Outcomes of free tissue transfer may be affected by vein grafts, which can be used for the placement of an autologous venous bypass or an arteriovenous (AV) loop leading to different hemodynamic situations. The purpose of this study was to investigate whether free flap anastomosis to a bypass or an AV loop can be performed with comparable results. Methods We performed a matched-pair analysis of 22 patients with PVD undergoing free flap reconstructions of the lower extremity with end-to-side anastomosis to an autologous venous bypass (n = 11, 1 female and 10 male patients) or an AV loop (n = 11, 2 female and 9 male patients). Defects caused by trauma, infection, ulcer, or fasciotomy were reconstructed in each group with 5 muscle-based flaps, 3 parascapular flaps, 2 anterolateral thigh (ALT) flaps, and 1 conjoined latissimus dorsi and parascapular flap. Postoperative complications including thromboses, flap failures, wound complications, and hematomas were compared. Results Postoperative complication rates including 1 venous pedicle thrombosis (9%vs. 0%, P = 1.0), minor (18% vs. 9%, P = 1.0) and major wound complications (45% vs. 27%, P = .69) as well as hematomas (27% vs. 36%, P = 1.0) did not show relevant differences between the groups. Flap failures were absent in both groups. Conclusion In patients with PVD, autologous venous bypass grafts may be used for end-to-side anastomoses of free flaps, with postoperative outcomes being comparable to AV loop reconstructions.

Background The placement of arteriovenous loops can enable microvascular anastomoses of free flaps when recipient vessels are scarce. In animal models, elevated fluid shear stress in arteriovenous loops promotes neoangiogenesis. Anecdotal reports in patients indicate that vein grafts used in free flap reconstructions of ischemic lower extremities are able to induce capillary formation. However, flow-stimulated angiogenesis has never been systematically investigated in humans, and it is unclear whether shear stress alters proangiogenic signaling pathways within the vascular wall of human arteriovenous loops. Methods Eight patients with lower extremity soft-tissue defects underwent two-stage reconstruction with arteriovenous loop placement, and free flap anastomoses to the loops 10 to 14 days later. Micro-RNA (miRNA) and gene expression profiles were determined in tissue samples harvested from vein grafts of arteriovenous loops by microarray analysis and quantitative real-time polymerase chain reaction. Samples from untreated veins served as controls. Results A strong deregulation of miRNA and gene expression was detected in arteriovenous loops, showing an overexpression of angiopoietic cytokines, oxygenation-associated genes, vascular growth factors, and connexin-43. The authors discovered inverse correlations along with validated and bioinformatically predicted interactions between angiogenesis-regulating genes and miRNAs in arteriovenous loops. Conclusions The authors' findings demonstrate that elevated shear stress triggers proangiogenic signaling pathways in human venous tissue, indicating that arteriovenous loops may have the ability to induce neoangiogenesis in humans. The authors' data corroborate the nutrient flap hypothesis and provide a molecular background for arteriovenous loop-based tissue engineering with potential clinical applications for soft-tissue defect reconstruction.

Restoration of adequate oral competence is especially challenging in double free flap reconstructions of massive head and neck defects resulting from composite resections. Our report illustrates that oral competence in double free flap reconstructions of extensive oromandibular defects can be successfully restored with tensor fascia lata suspension slings.

Soft tissue sarcomas occur most commonly in the lower and upper extremities. The standard treatment is limb salvage surgery combined with radiotherapy. Postoperative radiotherapy is associated with wound complications. This systematic review aims to summarise the available evidence and review the literature of the last 10 years regarding postoperative wound complications in patients who had limb salvage surgical excision followed by direct closure vs flap coverage together with postoperative radiotherapy and to define the optimal timeframe for adjuvant radiotherapy after soft tissue sarcomas resection and flap reconstruction. A literature search was performed using PubMed. The following keywords were searched: limb salvage, limb-sparing, flaps, radiation therapy, radiation, irradiation, adjuvant radiotherapy, postoperative radiotherapy, radiation effects, wound healing, surgical wound infection, surgical wound dehiscence, wound healing, soft tissue sarcoma and neoplasms. In total, 1045 papers were retrieved. Thirty-seven articles were finally selected after screening of abstracts and applying dates and language filters and inclusion and exclusion criteria. Plastic surgery provides a vast number of reconstructive flap procedures that are directly linked to decreasing wound complications, especially with the expectant postoperative radiotherapy. This adjuvant radiotherapy is better administered in the first 3-6 weeks after reconstruction to allow timely wound healing and avoid local recurrence.

Autologous free flaps are the criterion standard for reconstructions of complex soft tissue defects; however, they are limited by donor-site morbidities. The arteriovenous (AV) loop model enables the generation of soft tissue constructs based on acellular dermal matrices with a functional microvasculature and minimal donor site morbidity. The ideal scaffold for AV loop-based tissue engineering has not been determined.AV loops were placed into subcutaneous isolation chambers filled with either a collagen-elastin scaffold or a collagen-glycosaminoglycan scaffold in the thighs of rats. Matrix elasticity, neoangiogenesis, cell migration, and proliferation were compared after 14 and 28 days.Mean vessel count and area had increased in both matrices at 28 compared with 14 days. Collagen-elastin matrices showed a higher mean vessel count and area compared with collagen-glycosaminoglycan matrices at 14 days. At 28 days, a more homogeneous vascular network and higher cell counts were observed in collagen-elastin matrices. Collagen-glycosaminoglycan matrices, however, exhibited less volume loss at day 28.Collagen-based scaffolds are suitable for soft tissue engineering in conjunction with the AV loop technique. These scaffolds exhibit distinct patterns of angiogenesis, cell migration, and proliferation and may in the future serve as the basis of tissue-engineered free flaps as an individualized treatment concept for critical wounds.

The pathogenesis of aortic dilatation in patients with congenital aortic valve anomalies is poorly understood. Recent studies suggest that alterations of gene expression may be related to ascending aortic aneurysm formation in these patients. Knockout of endothelial nitric oxide synthase (eNOS) and GATA5 is associated with bicuspid aortic valves in mice. To study the role of eNOS and GATA5 in human congenital aortic valve disease and aortic dilatation, we investigated their gene expression in aortic tissue from patients with unicuspid, bicuspid, and tricuspid aortic valves.Samples from 84 patients (33 tricuspid, 32 bicuspid, and 19 unicuspid) were harvested intraoperatively from the ascending aorta. GATA5 and eNOS expression was determined by real-time polymerase chain reaction.GATA5 and eNOS expression in the aortic wall from patients with unicuspid aortic valves (GATA5: mean [M], 2.14; standard deviation [SD], 1.72; eNOS: M, 3.40; SD, 3.83) was significantly higher than in tricuspid aortic valves (GATA5: M, 1.12; SD, 0.80; eNOS: M, 1.00; SD, 0.74; each p0.05). Patients with bicuspid aortic valves (GATA5: M, 1.29, SD, 1.33; eNOS: M, 1.66; SD, 1.31) had a significantly higher eNOS expression than patients with tricuspid aortic valves (p0.05). The expression levels of eNOS and GATA5 correlated positively with each other and negatively with the ascending aortic diameter.Our data suggest that GATA5, possibly through upregulation of eNOS, plays a role in the development of aortic dilatation in patients with unicuspid and bicuspid aortic valves. The differential gene expression in patients with unicuspid compared with bicuspid aortic valves suggests that the pathogenesis of both aortic valve anomalies may be different.

The arteriovenous (AV) loop model permits the creation of significant volumes of axially vascularized tissue that represents an alternative to conventional free flaps, circumventing their common limitations. However, such AV-loop-based flaps have never before been examined in standardized animal models with respect to their suitability for reconstruction of critical bone exposing defects. In the course of our preliminary studies, we implemented a novel defect model in rats that provides standardized and critical wound conditions and evaluated whether AV-loop-generated flaps are suitable for free microsurgical transfer and closure of composite defects. We compared three groups of rodents with similar scapular defects – one received the AV-flap, whereas controls were left to heal by secondary intention or with supplementary acellular matrix alone. To create the flaps AV-loops were placed into subcutaneous teflon chambers filled with acellular matrix and transferred to the thigh region. Flap maturation was evaluated by histological analysis of angiogenesis and cell migration at days 14 and 28 after loop creation. Flap transfer to the scapular region and microsurgical anastomoses were performed after 14 days. Postoperative defect closure and perfusion were continually compared between groups. Within the AV-flap chamber mean vessel number, cell count, and the proportion of proliferating cells increased significantly over time. The novel defect model revealed that stable wound coverage with homogeneous vascular integration was achieved by AV-loop-vascularized soft tissue free flaps compared to controls. In summary, our study indicates for the first time that complex composite defects in rats can successfully be treated with AV-loop-based free flaps. This article is protected by copyright. All rights reserved.