Evaluation of the Effect of Botulinum Toxin A on the Lymphatic Endothelial Cells.

INTRODUCTION: Botulinum toxin A (BoTA) is a neurotoxin formed by Clostridium botulinum, with a broad medical application spectrum. While the primary effect of BoTA is on the muscles, the effects of BoTA in other systems including the blood vasculature have already been examined, revealing unexpected actions. However, no studies exist to the best of our knowledge regarding the potential effects of BoTA on the lymphatic vascular system, possessing a critical role in health and disease. Isolated human lymphatic endothelial cells (LECs) were cultured in dedicated in vitro culture systems. The analysis including imaging and cell culture approaches as well as molecular biology techniques is performed to examine the LEC alterations occurring upon exposure to different concentrations of BoTA. MATERIALS AND METHODS: Human LECs were cultured and expanded on collagen-coated petri dishes using endothelial basal medium and the commercial product Botox from Allergan as used for all our experiments. Harvested cells were used in various in vitro functional tests to assess the morphologic and functional properties of the BoTA-treated LECs. Gene expression analysis was performed to assess the most important lymphatic system-related genes and pathways. RESULTS: Concentrations of 1, 5 or 10 U of BoTA did not demonstrate a significant effect regarding the proliferation and migration capacity of the LECs versus untreated controls. Interestingly, even the smallest BoTA dose was found to significantly decrease the cord-like-structure formation capacity of the seeded LECs. Gene expression analysis was used to underpin possible molecular alterations, suggesting no significant effect of BoTA in the modification of gene expression versus the starvation medium control. CONCLUSION: LECs appear largely unaffected to BoTA treatment, with an isolated effect on the cord-like-structure formation capacity. Further work needs to assess the effect of BoTA on the smooth-muscle-cell-covered collecting lymphatic vessels and the possible aesthetic implications of such an effect, due to edema formation. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Characterization of the soft-tissue wall lining residual periodontal pockets and implications in periodontal wound healing.

AIM: To characterize the soft-tissue wall of remaining periodontal pockets for wound healing-related parameters versus healthy gingival crevices in the same individuals. MATERIALS AND METHODS: Gingival tissues collected from the diseased interface of pockets (GT biopsies) and from healthy gingival crevices (G biopsies) were subjected to RT2-profiler PCR Array for wound healing-related markers and network analysis of differentially expressed genes. Lymphangiogenesis-related gene expression was determined by qRT-PCR. The migration potential of mesenchymal stem cells isolated from GT biopsies (GT-MSCs) and G biopsies (G-MSCs) was evaluated by the scratch- and the transwell migration assays. The total collagen protein content was determined in GT-MSCs and G-MSCs homogenates. RESULTS: Gene-ontology analysis on significantly upregulated genes expressed in GT biopsies revealed enrichment of several genes involved in processes related to matrix remodeling, collagen deposition, and integrin signaling. No significantly expressed genes were seen in G biopsies. Regarding lymphangiogenesis-related genes, GT biopsies demonstrated greater expression for PROX1 than G biopsies (p = 0.05). Lower migration potential (p < 0.001), yet greater production of collagen protein (p = 0.05), was found for GT-MSCs over G-MSCs. CONCLUSION: Differential expression patterns of various molecular pathways in biopsies and cell cultures of diseased versus healthy gingival tissues indicate a potential of the former for tissue remodeling and repair. CLINICAL RELEVANCE: In the course of periodontitis, granulation tissue is formed within a periodontal defect in an attempt to reconstruct the site. Following treatment procedures periodontal granulation tissue remains inflamed but appears to retain healing potential.

A Comparative Analysis to Dissect the Histological and Molecular Differences among Lipedema, Lipohypertrophy and Secondary Lymphedema.

Lipedema, lipohypertrophy and secondary lymphedema are three conditions characterized by disproportionate subcutaneous fat accumulation affecting the extremities. Despite the apparent similarities and differences among their phenotypes, a comprehensive histological and molecular comparison does not yet exist, supporting the idea that there is an insufficient understanding of the conditions and particularly of lipohypertrophy. In our study, we performed histological and molecular analysis in anatomically-, BMI- and gender-matched samples of lipedema, lipohypertrophy and secondary lymphedema versus healthy control patients. Hereby, we found a significantly increased epidermal thickness only in patients with lipedema and secondary lymphedema, while significant adipocyte hypertrophy was identified in both lipedema and lipohypertrophy. Interestingly, the assessment of lymphatic vessel morphology showed significantly decreased total area coverage in lipohypertrophy versus the other conditions, while VEGF-D expression was significantly decreased across all conditions. The analysis of junctional genes often associated with permeability indicated a distinct and higher expression only in secondary lymphedema. Finally, the evaluation of the immune cell infiltrate verified the increased CD4+ cell and macrophage infiltration in lymphedema and lipedema respectively, without depicting a distinct immune cell profile in lipohypertrophy. Our study describes the distinct histological and molecular characteristics of lipohypertrophy, clearly distinguishing it from its two most important differential diagnoses.

A Distinct Cytokine Profile and Stromal Vascular Fraction Metabolic Status without Significant Changes in the Lipid Composition Characterizes Lipedema.

Lipedema is an adipose tissue disorder characterized by the disproportionate increase of subcutaneous fat tissue in the lower and/or upper extremities. The underlying pathomechanism remains unclear and no molecular biomarkers to distinguish the disease exist, leading to a large number of undiagnosed and misdiagnosed patients. To unravel the distinct molecular characteristic of lipedema we performed lipidomic analysis of the adipose tissue and serum of lipedema versus anatomically- and body mass index (BMI)-matched control patients. Both tissue groups showed no significant changes regarding lipid composition. As hyperplastic adipose tissue represents low-grade inflammation, the potential systemic effects on circulating cytokines were evaluated in lipedema and control patients using the Multiplex immunoassay system. Interestingly, increased systemic levels of interleukin 11 (p = 0.03), interleukin 28A (p = 0.04) and interleukin 29 (p = 0.04) were observed. As cytokines can influence metabolic activity, the metabolic phenotype of the stromal vascular fraction was examined, revealing significantly increased mitochondrial respiration in lipedema. In conclusion, despite sharing a comparable lipid profile with healthy adipose tissue, lipedema is characterized by a distinct systemic cytokine profile and metabolic activity of the stromal vascular fraction.

Adipose Tissue Hypertrophy, An Aberrant Biochemical Profile and Distinct Gene Expression in Lipedema.

BACKGROUND: Lipedema is a common adipose tissue disorder affecting women, characterized by a symmetric subcutaneous adipose tissue deposition, particularly of the lower extremities. Lipedema is usually underdiagnosed, thus remaining an undertreated disease. Importantly, no histopathologic or molecular hallmarks exist to clearly diagnose the disease, which is often misinterpreted as obesity or lymphedema. MATERIALS AND METHODS: The aim of the present study is to characterize in detail morphologic and molecular alterations in the adipose tissue composition of lipedema patients compared with healthy controls. Detailed histopathologic and molecular characterization was performed using lipid and cytokine quantification as well as gene expression arrays. The analysis was conducted on anatomically matched skin and fat tissue biopsies as well as fasting serum probes obtained from 10 lipedema and 11 gender and body mass index-matched control patients. RESULTS: Histologic evaluation of the adipose tissue showed increased intercellular fibrosis and adipocyte hypertrophy. Serum analysis showed an aberrant lipid metabolism without changes in the circulating adipokines. In an adipogenesis gene array, a distinct gene expression profile associated with macrophages was observed. Histologic assessment of the immune cell infiltrate confirmed the increased presence of macrophages, without changes in the T-cell compartment. CONCLUSIONS: Lipedema presents a distinguishable disease with typical tissue architecture and aberrant lipid metabolism, different to obesity or lymphedema. The differentially expressed genes and immune cell infiltration profile in lipedema patients further support these findings.

Prominent Lymphatic Vessel Hyperplasia with Progressive Dysfunction and Distinct Immune Cell Infiltration in Lymphedema.

Lymphedema is a common complication that occurs after breast cancer treatment in up to 30% of the patients undergoing surgical lymph node excision. It is associated with tissue swelling, fibrosis, increased risk of infection, and impaired wound healing. Despite the pronounced clinical manifestations of the disease, little is known about the morphological and functional characteristics of the lymphatic vasculature during the course of lymphedema progression. We used an experimental murine tail lymphedema model where sustained fluid stasis was generated on disruption of lymphatic flow, resulting in chronic edema formation with fibrosis and adipose tissue deposition. Morphological analysis of the lymphatic vessels revealed a dramatic expansion during the course of the disease, with active proliferation of lymphatic endothelial cells at the early stages of lymphedema. The lymphatic capillaries exhibited progressively impaired tracer filling and retrograde flow near the surgery site, whereas the collecting lymphatic vessels showed a gradually decreasing contraction amplitude with unchanged contraction frequency, leading to lymphatic contraction arrest at the later stages of the disease. Lymphedema onset was associated with pronounced infiltration by immune cells, predominantly Ly6G(+) and CD4(+) cells, which have been linked to impaired lymphatic vessel function.

High-Fat Diet in the Absence of Obesity Does Not Aggravate Surgically Induced Lymphoedema in Mice.

BACKGROUND: Lymphoedema represents the cardinal manifestation of lymphatic dysfunction and is associated with expansion of the adipose tissue in the affected limb. In mice, high-fat diet (HFD)-induced obesity was associated with impaired collecting lymphatic vessel function, and adiposity aggravated surgery-induced lymphoedema in a mouse model. The aim of the current study was to investigate whether adiposity is necessary to impair lymphatic function or whether increased lipid exposure alone might be sufficient in a surgical lymphoedema model. METHODS: To investigate the role of increased lipid exposure in lymphoedema development we used a well-established mouse tail lymphoedema model. Female mice were subjected to a short-term (6 weeks) HFD, without development of obesity, before surgical induction of lymphedema. Lymphoedema was followed over a period of 6 weeks measuring oedema, evaluating tissue histology and lymphatic vascular function. RESULTS: HFD increased baseline angiogenesis and average lymphatic vessel size in comparison to the chow control group. Upon induction of lymphedema, HFD-treated mice did not exhibit aggravated oedema and no morphological differences were observed in the blood and lymphatic vasculature. Importantly, the levels of fibro-adipose tissue deposition were comparable between the 2 groups and lymphatic vessel function was not impaired as a result of the HFD. Although the net immune cell infiltration was comparable, the HFD group displayed an increased infiltration of macrophages, which exhibited an M2 polarization phenotype. CONCLUSIONS: These results indicate that increased adiposity rather than dietary influences determines predisposition to or severity of lymphedema.

Animal models in surgical lymphedema research--a systematic review.

BACKGROUND: Chronic secondary lymphedema is a well-known complication in oncologic surgery. Autologous lymph node transplantation, lymphovenous anastomosis, and other lymphatic surgeries have been developed in the last decades with rising clinical application. Animal models to explore the pathophysiology of lymphedema and microsurgical interventions have reached great popularity, although the induction of stable lymphedema in animals is still challenging. The aim of this review was to systematically assess lymphedema animal models and their potential use to study surgical interventions. MATERIALS AND METHODS: A systematic review according to the PRISMA guidelines was performed without time or language restriction. Studies describing new or partially new models were included in chronological order. Models for primary and secondary lymphedema were assessed, and their potential for surgical procedures was evaluated. RESULTS: The systematic search yielded 8590 discrete articles. Of 180 articles included on basis of title, 84 were excluded after abstract review. Ninety-six were included in the final analysis with 24 key articles. CONCLUSIONS: No animal model is perfect, and many models show spontaneous lymphedema resolution. The rodent limb appears to be the most eligible animal model for experimental reconstruction of the lymphatic function as it is well accessible for vascularized tissue transfer. There is a need for standardized parameters in experimental lymphedema quantification. Also, more permanent models to study the effect of free vascularized lymph node transfer are needed.

Analysis of different outcome parameters and quality of life after different techniques of free vascularized lymph node transfer.

OBJECTIVE: Vascularized lymph node transfer (VLNT) has become an important surgical technique in the treatment of lymphedema. Considering the different available regions available for flap harvest, we aimed to analyze different donor sites for VLNT with respect to donor site morbidity, impact on limb volume, and patient-reported outcome measurements (PROMs). METHODS: A single-center prospective study of all patients undergoing VLNT at the Department of Plastic Surgery and Hand Surgery of the University Hospital Zurich between September 2016 and 2023 was conducted. Lymph nodes were harvested either from the omentum (gastroepiploic [GE]-VLNT), the lateral thoracic wall (LTW), or the superficial inguinal region (SI-VLNT). Volume measurements and PROMs were assessed preoperatively and at different postoperative intervals. RESULTS: Overall, 70 patients with upper limb lymphedema (21%) or lower limb lymphedema (79%) with different lymphedema stages were included. There were 49 patients who underwent GE-VLNT, followed by LTW-VLNT (n = 16) and SI-VLNT (n = 5). Lymph node harvest from the SI was associated with a significantly higher frequency of seroma development. The average percentage volume loss related in comparison to the preoperative volume of the affected limb was 9% after GE-VLNT, 10% after LTW-VLNT, and 5% after SI-VLNT without a significant difference between the groups. PROMs revealed significant improvements for physical functioning, symptoms and psychological well-being, with no differences between the VLNT techniques. CONCLUSIONS: VLNT leads to a significant improvement of quality of life and can decrease limb volume effectively, regardless of the selection of donor site. GE-VLNT has become our flap of choice owing to its low donor site morbidity and its properties that allow a double transplantation while avoiding a second donor site.

CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

Robotic-assisted Lymphovenous Anastomosis of the Central Lymphatic System.

Background: Recent advances in robotic microsurgery have enabled the application of robotic technology in central lymphatic reconstruction. Although the use of microsurgical robots demands careful consideration of associated costs and potentially prolonged operating times, it may offer improved surgical approaches and enhanced accessibility to deeper anatomical structures such as the thoracic duct (TD). Methods: We report on successful reconstruction of the central lymphatic system using the Symani Surgical System in four patients with lesions of the central lymphatic system. The patients were of different age (range: 8 mo-60 y) and had variable conditions, including central conducting lymphatic anomaly and other rare anomalies of the central lymphatic pathways. Results: Depending on the underlying pathology, a cervical access (n = 1) or median laparotomy (n = 3) was chosen to access the TD and perform anastomosis with a nearby vein. In all patients, anastomoses were patent, and chyle leakage decreased postoperatively. From a surgical perspective, the Symani Surgical System improved the precision of the microsurgeon and accessibility to the deep-lying TD. Conclusion: Considering the high morbidity and rarity of pathologies of the central lymphatic system, robotic-assisted microsurgery holds substantial promise in expanding and improving the microsurgical treatment for central lymphatic anomalies.

PROMs after Lymphatic Reconstructive Surgery: Is There a Correlation between Volume Reduction and Quality of Life?

Patients with upper limb lymphedema and lower limb lymphedema experience a wide range of physical and psychological symptoms that affect quality of life. The benefits of lymphatic reconstructive surgery for patients with lymphedema are undisputed. However, recording volume reduction alone may be insufficient with regard to postoperative outcome because measurements are often inadequate, depend on many factors, and do not reflect improvement in quality of life. Methods: We conducted a prospective single center study patients receiving lymphatic reconstructive surgery. Patients received volume measurements preoperatively and at standardized postoperative intervals. To evaluate patient-reported outcomes, patients completed the following questionnaires: LYMPH-Q Upper Extremity Module, quickDASH, SF 36, Lymphoedema Functioning, Disability and Health Questionnaire for Lower Limb Lymphoedema, and Lower Extremity Functional Scale at the aforementioned intervals. Results: We included 55 patients with upper limb lymphedema (24%) and lower limb lymphedema (73%) of lymphedema grades I-III. Patients received lymphovenous anastomosis only (23%), free vascularized lymph node transfer (35%) or a combination of both (42%). Analysis of patient-reported outcome measurements revealed improvements with respect to a broad range of complaints, particularly physical function, symptoms, and psychological well-being. There was no correlation between the extent of volume reduction and improvement in quality of life (Pearson correlation coefficient below ±0.7; P > 0.05). Conclusions: Based on a broad range of outcome measurements, we observed an improved quality of life in almost all patients, even in those without measurable volume loss of the extremity operated on, which emphasizes the need for a standardized use of patient-reported outcome measures to evaluate the benefits of lymphatic reconstructive surgery.

[Robotics in plastic surgery]. / Robotik in der plastischen Chirurgie.

In recent years surgical robotic systems which were specifically developed for microsurgery have expanded the application of robotic-assisted surgery to plastic reconstructive surgery. Currently, there are two microsurgical robotic systems available for reconstructive plastic surgery. Both systems feature tremor reduction and motion scaling technologies, which are intended to optimize the surgeon's precision and dexterity. In the Department of Plastic Surgery and Hand Surgery at the University Hospital Zurich, the Symani Surgical System® has already been used for many microsurgical and supermicrosurgical operations, including autologous breast reconstruction, nerve transfer and, in particular, reconstructive lymphatic surgery. Despite special technical challenges, such as a lack of haptic feedback, the advantages outweigh the disadvantages for an appropriately trained and skilled microsurgeon, including smaller surgical access incisions for anatomically deep structures and an improvement in surgical precision.

Benefits of robotic-assisted lymphatic microsurgery in deep anatomical planes.

Micro- and supermicrosurgeries have become standard techniques for lymphatic reconstruction. As increasingly smaller vessels are being targeted, robotic-assisted surgery has emerged as a new approach to push reconstructive limits owing to its ability of motion scaling and providing better accessibility of deep anatomical regions. The precision of the robot is achieved at the expense of operating speed among other variables; therefore, the surgeon must weigh the enhanced dexterity against the additional operating time and cost required for the robotic surgical system itself to ensure optimal resource utilization. Here we present a case series of 8 patients who underwent robot-assisted lymphatic microsurgery for omental flap transfer to the axilla and lympho-venous anastomosis. The Symani® Surgical System was used with a conventional microscope or 3D exoscope. The use of 3D exoscope provided clear benefits in terms of surgeon positioning. Moreover, access to the recipient vessels near the thoracic wall was significantly improved with the robotic setup. In addition, suture precision was excellent, resulting in patent anastomoses. Operating time for anastomosis was comparable to that for manual anastomosis and demonstrated a steep learning curve. The benefits of robotic systems in operating fields with good exposure require further evaluation. However, owing to longer instruments, additional stability, dexterity, and motion precision, robotic systems offer a marked advantage for operating in deep anatomical planes and on small structures. A potentially new field for the implementation of robotic surgery is central lymphatic reconstruction. Progress in terms of operating time and cost is crucial, and future research should validate the effectiveness of robotic-assisted surgery in larger clinical studies.

Outcome of Facial Burn Injuries Treated by a Nanofibrous Temporary Epidermal Layer.

BACKGROUND: The face is commonly affected in thermal injuries, with a demand for proper recognition and the correct choice of treatment to guarantee optimal aesthetic and functional outcomes. It is highly vascularized and often heals conservatively, highlighting the particular relevance of conservative treatment modalities, many of which require daily re-applications or dressing changes, which can be painful and tedious for both the patient and the healthcare providers. Motivated by encouraging results of a novel temporary nanofibrous epidermal layer, we herein present a case series of this technology in a case series of patients suffering from facial burns and treated in our Burn Center. PATIENTS AND METHODS: Patients with superficial partial-thickness facial burns and mixed pattern burns, which were treated with SpinCare™, an electrospun nanofibrous temporary epidermal layer, between 2019 and 2021, at our institution were analyzed retrospectively. The Manchester scar scale (MSS) and numeric rating scale (NRS) were used for scar, pain, and outcome evaluation at different time points by five independent board-certified plastic surgeons with profound experience in burn surgery. RESULTS: Ten patients (m = 9; f = 1) were treated and evaluated retrospectively. The mean age was 38.8 ± years (SD ± 17.85). The mean healing time was 6.4 days (SD ± 1.56). The mean follow-up was 16.4 months (SD ± 11.33). The mean MSS score was 5.06 (SD ± 1.31), and the mean NRS Score for pain was significantly reduced from initially 7 to 0.875 upon application (mean (pre-application) 7 ± 0.7 and (application) 0.875 ± 1.26; p ≤ 0.0001). Patients reported a NRS score of 10 in terms of functional and cosmetic outcomes at their final follow-up appointment. No adverse effects were observed. CONCLUSIONS: The application of a nanofibrous temporary epidermal layer such as SpinCare™ represents a relatively easy-to-use, well-tolerated, and effective alternative for the treatment of partial-thickness facial burns.

Involvement of the Macrophage Migration Inhibitory Factor (MIF) in Lipedema.

Lipedema is a chronic disorder that mainly affects women. It is often misdiagnosed, and its etiology remains unknown. Recent research indicates an accumulation of macrophages and a shift in macrophage polarization in lipedema. One known protein superfamily that contributes to macrophage accumulation and polarization is the macrophage migration inhibitory factor (MIF) family. MIF-1 and MIF-2 are ubiquitously expressed and also regulate inflammatory processes in adipose tissue. In this study, the expression of MIF-1, MIF-2 and CD74-a common receptor for both cytokines-was analyzed in tissue samples of 11 lipedema and 11 BMI-matched, age-matched and anatomically matched control patients using qPCR and immunohistochemistry (IHC). The mRNA expression of MIF-1 (mean 1.256; SD 0.303; p = 0.0485) and CD74 (mean 1.514; SD 0.397; p = 0.0097) were significantly elevated in lipedema patients, while MIF-2 expression was unaffected (mean 1.004; SD 0.358; p = 0.9718). The IHC analysis corroborated the results for CD74 expression on a cellular level. In conclusion, our results provide first evidence for a potential involvement of the MIF family, presumably via the MIF-1-CD74 axis, in lipedema.

Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator.

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

Early Experience Using a New Robotic Microsurgical System for Lymphatic Surgery.

Robotic microsurgery has emerged as a new technology with potential benefits for reconstructive surgery. We report the first-in-human use of the Symani surgical system to perform lympho-venous and arterial anastomosis for lymphatic reconstruction. In five patients, 10 robot-assisted anastomoses were performed. Next to lympho-venous anastomoses, two patients received a free vascularized lymph node transfer. Motion scaling was set to 10×. Visualization was either achieved with a 3D system or an optical microscope. All anastomoses were patent as confirmed by ICG. Despite a longer time to perform the first anastomoses with the robot, we observed a decline in duration of anastomosis. Among the advantages of the system were a high accuracy in placing the stitches even in very small and fragile vessels or when performing anastomoses with size mismatches. The challenges encountered included the lack of a touch sensation and the necessity to develop a "see-feel." This could be achieved surprisingly well because the force necessary to close dilator and needle holder via the manipulators was perceived as comparable to using conventional micro instruments. Our data confirm feasibility and safety of the robotic system to perform lymphatic surgery. Larger patient cohorts and inclusion of surgeons at different training levels will be necessary to investigate the true potential of robotics in microsurgery. In addition, robot-assisted surgery shows a promising potential in opening up new frontiers in reconstructive microsurgery (eg, the reliable performance of anastomoses on even smaller blood and lymphatic vessels or on structures deeper within the body cavities-eg, the thoracic duct).

In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt.

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling.

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.