Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

Background: Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes. Methods: Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Results: Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Conclusion: Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA.

Approaches to the Management of Synkinesis: A Scoping Review.

Postparalysis facial synkinesis (PPFS) can develop in any facial palsy and is associated with significant functional and psychosocial consequences for affected patients. While the prevention of synkinesis especially after Bell's palsy has been well examined, much less evidence exists regarding the management of patients with already established synkinesis. Therefore, the purpose of this review is to summarize the available literature and to provide an overview of the current therapeutic options for facial palsy patients with established synkinesis. A systematic literature review was undertaken, following the Preferred Reporting Items of Systematic Reviews and Meta-analyses 2020 guidelines. MEDLINE via PubMed and Cochrane Library were searched using the following strategy: ([facial palsy] OR [facial paralysis] OR [facial paresis]) AND (synkinesis) AND ([management] OR [guidelines] OR [treatment]). The initial search yielded 201 articles of which 36 original papers and 2 meta-analyses met the criteria for inclusion. Overall, the included articles provided original outcome data on 1,408 patients. Articles were divided into the following treatment categories: chemodenervation (12 studies, 536 patients), facial therapy (5 studies, 206 patients), surgical (10 studies, 389 patients), and combination therapy (9 studies, 278 patients). Results are analyzed and discussed accordingly. Significant heterogeneity in study population and design, lack of control groups, differences in postoperative follow-up, as well as the use of a variety of subjective and objective assessment tools to quantify synkinesis prevent direct comparison between treatment modalities. To date, there is no consensus on how PPFS is best treated. The lack of comparative studies and standardized outcome reporting hinder our understanding of this complex condition. Until higher quality scientific evidence is available, it remains a challenge best approached in an interdisciplinary team. An individualized multimodal therapeutic concept consisting of facial therapy, chemodenervation, and surgery should be tailored to meet the specific needs of the patient.

How to Treat a Cyclist's Nodule?-Introduction of a Novel, ICG-Assisted Approach.

BACKGROUND: Perineal nodular induration (PNI) is a benign proliferation of the soft tissue in the perineal region that is associated with saddle sports, especially road cycling. The etiology has not been conclusively clarified; however, repeated microtrauma to the collagen and subcutaneous fat tissue by pressure, vibration and shear forces is considered a mechanical pathomechanism. In this context, chronic lymphedema resulting in the development of fibrous tissue has been suggested as an etiological pathway of PNI. The primary aim of this study was to introduce and elucidate a novel operative technique regarding PNI that is assisted by indocyanine green (ICG). In order to provide some context for this approach, we conducted a comprehensive review of the existing literature. This dual objective aimed to contribute to the existing body of knowledge while introducing an innovative surgical approach for managing PNI. METHODS: We reviewed publications relating to PNI published between 1990 and 2023. In addition to the thorough review of the literature, we presented our novel surgical approach. We described how this elaborate approach for extensive cases of PNI involves surgical excision combined with tissue doubling and intraoperative ICG visualization for exact lymphatic vessel obliteration to minimize the risk of recurrence based on the presumed context of lymphatic congestion. RESULTS: The literature research yielded 16 PubMed articles encompassing 23 cases of perineal nodular induration (PNI) or cyclist's nodule. Of these, 9 cases involved females, and 14 involved males. Conservative treatment was documented in 7 cases (30%), while surgical approaches were reported in 16 cases (70%). Notably, a limited number of articles focused on histopathological or radiological characteristics, with a shortage of structured reviews on surgical treatment options. Only two articles provided detailed insights into surgical techniques. Similarly to the two cases of surgical intervention identified in the literature research, the post-operative recovery in our ICG assisted surgical approach was prompt, meaning a return to cycling was possible six weeks after surgery. At the end of the observation period (twelve months after surgery), regular scar formation and no signs of recurrence were seen. CONCLUSION: We hope that this article draws attention to the condition of PNI in times of increasing popularity of cycling as a sport. We aimed to contribute to the existing body of knowledge through our thorough review of the existing literature while introducing an innovative surgical approach for managing PNI. Due to the successful outcome, the combination of tissue doubling, intraoperative ICG visualization and postoperative negative wound therapy should be considered as a therapeutic strategy in cases of large PNI.

Mid-term Clinical Outcome of Microvascular Gracilis Muscle Flaps for Defects of the Hand.

PURPOSE: Gracilis muscle flaps are useful to cover defects of the hand. However, there are currently no studies describing outcome measurements after covering soft tissue defects using free flaps in the hand. AIM: To analyze mid-term results of gracilis muscle flap coverage for defects on the hand, with regard to functional and esthetic integrity. METHODS: 16 patients aged 44.3 (range 20-70) years were re-examined after a mean follow-up of 23.6 (range 2-77) months. Mean defect size was 124 (range 52-300) cm2 located palmar (n = 9), dorsal (n = 6), or radial (n = 1). All flaps were performed as microvascular muscle flaps, covered by split thickness skin graft. RESULTS: Flaps survived in 15 patients. 6 patients required reoperations. Reasons for revisions were venous anastomosis failure with total flap loss (n = 1) requiring a second gracilis muscle flap; necrosis at the tip of the flap (n = 1) with renewed split thickness skin cover. A surplus of the flap (n = 2) required flap thinning and scar corrections were performed in 2 patients. Mean grip strength was 25% (range 33.3-96.4%) compared to the contralateral side and mean patient-reported satisfaction 1.4 (range 1-3) (1 = excellent; 4 = poor). CONCLUSIONS: Gracilis muscle flaps showed a survival rate of 94%. Patients showed good clinical outcomes with acceptable wrist movements and grip strength as well as high reported satisfaction rates. Compared to fasciocutaneous free flaps, pliability and thinness especially on the palmar aspect of the hand are advantageous. Hence, covering large defects of the hand with a gracilis muscle flap can be a very satisfactory procedure. LEVEL OF EVIDENCE: IV observational.

Challenges and opportunities in vascularized composite allotransplantation of joints: a systematic literature review.

Background: Joint allotransplantation (JA) within the field of vascularized composite allotransplantation (VCA) holds great potential for functional and non-prosthetic reconstruction of severely damaged joints. However, clinical use of JA remains limited due to the immune rejection associated with all forms of allotransplantation. In this study, we aim to provide a comprehensive overview of the current state of JA through a systematic review of clinical, animal, and immunological studies on this topic. Methods: We conducted a systematic literature review in accordance with the PRISMA guidelines to identify relevant articles in PubMed, Cochrane Library, and Web of Science databases. The results were analyzed, and potential future prospects were discussed in detail. Results: Our review included 14 articles describing relevant developments in JA. Currently, most JA-related research is being performed in small animal models, demonstrating graft survival and functional restoration with short-term immunosuppression. In human patients, only six knee allotransplantations have been performed to date, with all grafts ultimately failing and a maximum graft survival of 56 months. Conclusion: Research on joint allotransplantation has been limited over the last 20 years due to the rarity of clinical applications, the complex nature of surgical procedures, and uncertain outcomes stemming from immune rejection. However, the key to overcoming these challenges lies in extending graft survival and minimizing immunosuppressive side effects. With the emergence of new immunosuppressive strategies, the feasibility and clinical potential of vascularized joint allotransplantation warrants further investigation.

The osteocutaneous SCIP flap: A detailed description of the surgical technique and retrospective cohort study of consecutive cases in a tertiary European centre.

INTRODUCTION: In the era of increasing popularity of the superficial circumflex iliac perforator (SCIP) flap, osteocutaneous variants of the flap have been described as well. Despite their benefits such as customizability and low donor site morbidity, these flaps have not yet gained broad acceptance. By reviewing our case series, we aim to promote the safe application of this promising new tool in osteoplastic reconstructions. PATIENTS AND METHODS: We performed a single-centre, retrospective chart review of all cases in which osteocutaneous SCIP-flaps were used. We describe our surgical technique and present the surgical, functional and aesthetic outcomes of the patients in our cohort. RESULTS: Since September 2019, we have used osteocutaneous SCIP flaps in six patients, five in the extremities and one for the head and neck region. The vascularised bone segment was measured on average 4.9 cm (range 4-7 cm) x 3 cm (range 1.5-4 cm) and was combined with a skin paddle of a mean length of 14.3 cm (range 8-20 cm) and width of 6.3 cm (range 5-8 cm). One flap underwent emergency revision due to venous congestion. All flaps survived and healed uneventfully. Long-term follow-up shows adequate bony integration and stable soft tissue coverage with good functional restoration and minimal donor site morbidity. CONCLUSION: The osteocutaneous SCIP flap provides a large and thin skin island and a "moderately sized" vascularised bone segment with minimal donor site morbidity and can be successfully used in selected cases of osteoplastic reconstruction.

Transcutaneous sentinel lymph node detection in cutaneous melanoma with indocyanine green and near-infrared fluorescence: A diagnostic sensitivity study.

Sentinel lymph node (SLN) biopsy with preoperative radiocolloid-based lymphoscintigraphy and blue dye injection is considered the standard procedure for staging nodal metastases in early-stage cutaneous melanoma patients with clinically uninvolved lymph nodes. While this combination renders good accuracy in SLN detection, radiation exposure and the frequent allergic reactions to the blue dye are considered drawbacks of this technique. Indocyanine green (ICG) is a water-soluble fluorescent dye that can be identified through near-infrared fluorescence imaging (NIRFI). The aim of this prospective diagnostic sensitivity study was to assess the feasibility of ICG and NIRFI to identify SLNs in melanoma transcutaneously ("before skin incision") and to analyze the various factors influencing detection rate, in comparison to lymphoscintigraphy. This study included 93 patients undergoing SLN biopsy for cutaneous melanoma. The region and the number of the SLNs identified with lymphoscintigraphy and with ICG were recorded. Patients' characteristics, as well as tumor details were also recorded preoperatively. One hundred and ninety-four SLNs were identified through lymphoscintigraphy. The sensitivity of ICG for transcutaneous identification of the location of the SLNs was 96.1% overall, while the sensitivity rate for the number of SLNs was 79.4%. Gender and age did not seem to influence detection rate, but a body mass index >30 kg/m2 was associated with a lower identification rate of the number of SLNs (P = .045). Transcutaneous identification of SLNs through ICG and NIRFI technology is a feasible technique that could potentially replace in selected patients the standard SLN detection methodology in cutaneous melanoma.

The anatomical reliability of the superficial circumflex iliac artery perforator (SCIP) flap.

INTRODUCTION: In order to achieve a satisfactory functional and aesthetic result a thin skin flap is often required in surgical reconstruction of various body regions. Perforator flaps based on either the superficial or deep branch of the superficial circumflex iliac artery (SCIA) have been used for this purpose mainly in the Asian population. Recently the superficial plane has been established as a new way of elevating the flap. Anatomical studies and details of this new flap are lacking. MATERIAL AND METHODS: Wide areas were harvested subfascially from the groin of Thiel-fixated cadavers. Both deep and superficial branches of the superficial circumflex iliac artery were carefully dissected and individually injected with µAngiofil. After CT-imaging the flaps were raised on the superficial plane, perforators were marked and the flaps subsequently rescanned. High-resolution images of regions of interest were taken using micro-CT. RESULTS: A total of 21 flaps were harvested and analyzed. Both the deep and superficial branch provided more than three perforators per branch, however, the deep branch based flap was significantly larger (202 vs. 112 cm2, p < 0.01) and had a longer pedicle (9.1 vs. 6.6 cm, p < 0.01). Raising the flap in the superficial plane reliably reduces bulk and increases homogeneity. CONCLUSIONS: The SCIP flap appears to have a reliable vascular blood supply. The SCIA and its main branches and perforators have a consistent vascular pattern. The deep branch of the SCIA has the anatomic potential to be the preferred pedicle in case larger flaps with longer pedicles are necessary.

Development of vascularized nerve scaffold using perfusion-decellularization and recellularization.

INTRODUCTION: Vascularized nerve grafts (VNG) may offer an advantage in peripheral nerve regeneration by avoiding ischemic damage and central necrosis observed in non-VNG, particularly for the treatment of large and long nerve defects. However, surgical complexity, donor site morbidity and limited nerve availability remain important drawbacks for the clinical use of VNG. Here we explore the potential of perfusion-decellularization for bioengineering a VNG to be used in peripheral nerve reconstruction. METHODS: Porcine sciatic nerves were surgically procured along with their vascular pedicle attached. The specimens were decellularized via perfusion-decellularization and preservation of the extracellular matrix (ECM), vascular patency and tissue cytokine contents were examined. Scaffold reendothelialization was conducted with porcine aortic endothelial cells in a perfusion-bioreactor. RESULTS: Morphologic examination of decellularized VNG and analysis of the DNA content demonstrated cell clearance whereas ECM content and structures of the nerve fascicles were preserved. Using 3D micro-computed tomography imaging we observed optimal vasculature preservation in decellularized scaffolds, down to the capillary level. Cytokine quantification demonstrated measurable levels of growth factors after decellularization. Endothelial cell engraftment of the large caliber vessels was observed in reendothelialized scaffolds. CONCLUSIONS: In this study we provide evidence that perfusion-decellularization can be used to create vascularized nerve scaffolds in which the vasculature and the ECM component are well preserved. As compared to non-vascularized conduits, engineered vascularized nerve scaffolds may represent an ideal approach for promoting better nerve regeneration in larger nerve defect reconstructions.

Cutting-edge microangio-CT: new dimensions in vascular imaging and kidney morphometry.

In the last decades, the contrast-enhanced micro-computed tomography (micro-CT) imaging of a whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop micro-CT scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature and glomeruli number. This method is based on µAngiofil, a new polymerizing contrast agent with homogenous X-ray absorption, which provides continuous filling of the complete vasculature and enables correlative imaging approaches. For rapid and reliable kidney morphometry, the microangio-CT (µaCT) data sets from glial cell line-derived neurotrophic factor (GDNF)+/- mice and their wild-type littermates were used. The results were obtained much faster compared with the current gold standard, histology-based stereology, and without processing artifacts. The histology-based morphometry was done afterward on the same kidneys. Both approaches revealed that the GDNF+/- male mice had about 40% fewer glomeruli. Furthermore, our approach allows for the definition of sites of interest for further histological investigation, i.e., correlative morphology. The polymerized µAngiofil stays in perfused vessels and is autofluorescent, which is what greatly facilitates the matching of histological sections with µaCT data. The presented approach is a time-efficient, reliable, qualitative, and quantitative methodology. Besides glomerular morphometry, the µaCT data can be used for qualitative and quantitative analysis of the kidney vasculature and correlative morphology.