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.

Skin Color Match in Autologous Breast Reconstruction: Which Donor Site Gives the Best Result?

BACKGROUND: Color match of a reconstructed breast with the surrounding area is of importance for the overall aesthetic result. The objective of our study was to quantify the degree of color match achieved with different autologous breast reconstructions and to analyze the changes in color over time by analyzing digital photographs. METHODS: 193 patients that underwent a delayed autologous breast reconstruction (DIEP, PAP, LAP, LD) were included. Standardized pictures from 242 flaps at 3 months and 9-12 months postoperative were analyzed and the L*a*b* values and delta E2000 (dE) values were determined to qualify the color match. The Kruskal-Wallis and Wilcoxon rank-sum tests were used for statistical analysis. RESULTS: Initially, DIEP flaps had a significant lower dE value compared to LD (p=0.012) and PAP flaps (p < 0.001) when compared with the natural breast. PAP flaps showed a significant decrease after 9-12 months (p=0.003). Perception of color match was in all flaps comparable. Compared to the cleavage, at late follow-up, DIEP flaps had a significant higher dE value compared to LD (p=0.017) and PAP flaps (p < 0.001). PAP flaps presented a significant decrease of dE after 9-12 months (p =0.031). Abdominal skin presented no better skin color match in patients with PAP, LD, and LAP flaps. CONCLUSIONS: All analyzed flaps have a comparable color match with the surrounding tissue as well as with the contralateral breast about one year after surgery. The color of PAP flaps changes more, which leads to an improvement at a later follow-up.

Protein Aggregation on Metal Oxides Governs Catalytic Activity and Cellular Uptake.

Engineering of catalytically active inorganic nanomaterials holds promising prospects for biomedicine. Catalytically active metal oxides show applications in enhancing wound healing but have also been employed to induce cell death in photodynamic or radiation therapy. Upon introduction into a biological system, nanomaterials are exposed to complex fluids, causing interaction and adsorption of ions and proteins. While protein corona formation on nanomaterials is acknowledged, its modulation of nanomaterial catalytic efficacy is less understood. In this study, proteomic analyses and nano-analytic methodologies quantify and characterize adsorbed proteins, correlating this protein layer with metal oxide catalytic activity in vitro and in vivo. The protein corona comprises up to 280 different proteins, constituting up to 38% by weight. Enhanced complement factors and other opsonins on nanocatalyst surfaces lead to their uptake into macrophages when applied topically, localizing >99% of the nanomaterials in tissue-resident macrophages. Initially, the formation of the protein corona significantly reduces the nanocatalysts' activity, but this activity can be partially recovered in endosomal conditions due to the proteolytic degradation of the corona. Overall, the research reveals the complex relationship between physisorbed proteins and the catalytic characteristics of specific metal oxide nanoparticles, providing design parameters for optimizing nanocatalysts in complex biological environments.

Bioglass/ceria nanoparticle hybrids for the treatment of seroma: a comparative long-term study in rats.

Background: Seroma formation is a common postoperative complication. Fibrin-based glues are typically employed in an attempt to seal the cavity. Recently, the first nanoparticle (NP)-based treatment approaches have emerged. Nanoparticle dispersions can be used as tissue glues, capitalizing on a phenomenon known as 'nanobridging'. In this process, macromolecules such as proteins physically adsorb onto the NP surface, leading to macroscopic adhesion. Although significant early seroma reduction has been shown, little is known about long-term efficacy of NPs. The aim of this study was to assess the long-term effects of NPs in reducing seroma formation, and to understand their underlying mechanism. Methods: Seroma was surgically induced bilaterally in 20 Lewis rats. On postoperative day (POD) 7, seromas were aspirated on both sides. In 10 rats, one side was treated with NPs, while the contralateral side received only NP carrier solution. In the other 10 rats, one side was treated with fibrin glue, while the other was left untreated. Seroma fluid, blood and tissue samples were obtained at defined time points. Biochemical, histopathological and immunohistochemical assessments were made. Results: NP-treated sides showed no macroscopically visible seroma formation after application on POD 7, in stark contrast to the fibrin-treated sides, where 60% of the rats had seromas on POD 14, and 50% on POD 21. At the endpoint (POD 42), sides treated with nanoparticles (NPs) exhibited significant macroscopic differences compared to other groups, including the absence of a cavity, and increased fibrous adhesions. Histologically, there were more macrophage groupings and collagen type 1 (COL1) deposits in the superficial capsule on NP-treated sides. Conclusion: NPs not only significantly reduced early manifestations of seroma and demonstrated an anti-inflammatory response, but they also led to increased adhesion formation over the long term, suggesting a decreased risk of seroma recurrence. These findings highlight both the adhesive properties of NPs and their potential for clinical therapy.

Free Tissue Transfer in Head and Neck Reconstruction: A Multidisciplinary 15-Year Experience.

BACKGROUND: Free tissue transfer is considered the gold standard in reconstruction of extensive defects in head and neck surgery. The aim of this 15-year retrospective study is to analyze the outcomes of free tissue transfers in the head and neck area in a tertiary referral university hospital. MATERIALS AND METHODS: A retrospective, single-center study of all patients undergoing free tissue transfers for head and neck reconstruction between 2006 and 2020 was performed. Patient demographics, comorbidities, flap characteristics, outcomes and complications were assessed. RESULTS: A total of 353 free flaps were performed. The most common defect etiology was synchronous oncologic resection (74.2%). The majority of patients had at least one comorbidity (70.3%), with smoking recorded in 46.2% of the cases and alcohol consumption in 31.7%. The anterolateral thigh flap was the most commonly used flap (37.7%), followed by the osteoseptocutaneous fibula flap (26.9%). Our overall flap success rate was 97.7%, while the overall complication rate was 45.9%. CONCLUSIONS: Free tissue transfer in head and neck reconstruction is reliable. However, complication rates remain high due to the complexity of such cases and frequent presence of comorbidities. Nonetheless, when effectively managed within a multidisciplinary team, complications rarely jeopardize the overall reconstruction outcome.

Flap Reconstruction Outcome Following Surgical Resection of Soft Tissue and Bone Sarcoma in the Setting of (Neo)adjuvant Therapy: A Sarcoma Center Experience.

BACKGROUND: Soft tissue and bone sarcomas are heterogeneous groups of malignant tumors. The shift in their management, with an emphasis on limb salvage, has deemed the involvement of reconstructive surgeons an integral part of their multidisciplinary treatment. We present our experience with free and pedicled flaps in the reconstruction of sarcomas at a tertiary referral university hospital and major sarcoma center. MATERIALS AND METHODS: All patients undergoing flap reconstruction after sarcoma resection over a 5-year period have been included in the study. Patient-related data and postoperative complications were collected retrospectively, ensuring a minimum follow-up of 3 years. RESULTS: A total of 90 patients underwent treatment with 26 free flaps and 64 pedicled flaps. Postoperative complications occurred in 37.7% of patients, and the flap failure rate was 4.4%. Diabetes, alcohol consumption and male gender were associated with increased early necrosis of the flap. Preoperative chemotherapy significantly increased the occurrence of early infection and late dehiscence, while preoperative radiotherapy was associated with a higher incidence of lymphedema. Intraoperative radiotherapy was associated with late seromas and lymphedema. CONCLUSIONS: Reconstructive surgery with either pedicled or free flaps is reliable, but it can be demanding in the setting of sarcoma surgery. A higher complication rate is to be expected with neoadjuvant therapy and with certain comorbidities.

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.

Management of Postoperative Seroma: Recommendations Based on a 12-Year Retrospective Study.

Introduction: Seroma formation is a serious postoperative complication. Since the management algorithms available in the literature are scarce, we aimed to analyze our experience with postoperative seroma in order to identify indicators for revisional surgery and propose recommendations for management. Methods: This retrospective study included all patients with postoperative seroma treated in a tertiary university hospital from 2008 to 2020. Patients' demographics, medical history, and seroma treatment details were recorded and analyzed. Results: Overall, 156 patients were included: 41% were initially treated through needle aspiration, with 61% eventually undergoing surgical treatment for postoperative seroma. Comorbidities, such as heart failure and coronary heart disease, were significantly associated with an increased need for revisional surgery (p < 0.05). Both a duration of >40 days of repeated needle aspirations and drain re-insertions were significantly correlated with an increased risk for revisional surgery (p < 0.05). Conclusion: Patients requiring seroma aspiration should be counseled on surgical treatment sooner rather than later, as prolonged aspiration time (over 40 days) greatly increases the risk of surgical revision. Moreover, the reinsertion of a drain should only be used as a temporizing measure, at most, and patients requiring a drain to control the size of the seroma should promptly be scheduled for a surgical revision.

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.

Post-bariatric contour deformity correction: an endeavour to establish objective criteria nationally.

BACKGROUND: A marked increase in bariatric surgery has led to higher numbers of patients with contour deformities after massive weight loss seeking plastic surgical correction. Insurance coverage for these post-bariatric interventions is highly subjective and a common set of objective criteria has not yet been established. AIM: The aim was to evaluate the factors influencing insurance coverage for post-bariatric surgery, focusing on finding objective, reproducible criteria. METHODS: This was a retrospective single centre chart review of all post-bariatric patients with redundant skin requesting body contouring surgery from 2013 to 2018. Demographic, bariatric and surgical, as well as insurance information were collected. A logistic regression model was used to identify predictors of successful insurance coverage. RESULTS: 116 Patients were included in the study. Insurance approval for post-bariatric body contouring surgery was obtained for only 47 patients (41%). Mentioning the term "medical indication" in the application letter was associated with a 15.2 times higher rate of receiving a positive answer (p <0.001), whereas mentioning "mental suffering" was associated with 82.3% lower chance of getting a positive response (p <0.001). A high body mass index (BMI) (p <0.009) before the bariatric operation as well as a high BMI reduction (p <0.021) were associated with a higher approval rate by insurance companies . An additional application letter to the insurance company (p <0.024) as well as mentioning mechanical restriction (p <0.022) were associated with a positive response from the insurance companies. CONCLUSIONS: We were able to establish certain objective predictive criteria for insurance coverage of post-bariatric surgery. However, it appears that the decisions of insurance companies for this condition are still rather randomly taken. Therefore, the establishment of objective criteria for insurance coverage may allow fairer treatment for this growing patient population.

Lop Ear to Conchal Microtia: An Algorithmic Surgical Approach.

BACKGROUND: The lop ear deformity is defined by a deficient helix and scapha, underdeveloped anthelix, and downfolding of the helix. The terminology used is still confusing, and the treatment is not entirely structured. The aim of this study was to provide a new systematic surgical approach of this deformity based on our center's experience. MATERIALS AND METHODS: All patients undergoing surgical correction of lop ears between 2007 and 2019 at Great Ormond Street Hospital were included. Patients' data, surgical techniques, and postoperative complications were recorded. RESULTS: Based on our records, we identified 3 surgical techniques for the correction of lop ears, based on the degree of deformity encountered. In a mild lop ear, correction was achieved with a modified otoplasty technique by improving the definition of the antihelix and superior crus. In a moderate deformity, additional remodeling of the lidded helix was performed (extended otoplasty), whereas for the severe lop ear, the amount of cupping and the deficient cartilage required formal reconstruction using a carved rib cartilage framework. There were a total of 109 patients and 146 lop ears: 58 mild, 27 moderate, and 61 severe lop ears. CONCLUSION: We feel that there is a point in the spectrum of congenital ear deformity when a severe lop ear becomes a conchal microtia and recommend this approach to simplify the management of these cases. This is intended to bring greater clarity to how to deal with lop ears, based on the severity of the deformity and the surgical techniques used.

Presence of Donor Lymph Nodes Within Vascularized Composite Allotransplantation Ameliorates VEGF-C-mediated Lymphangiogenesis and Delays the Onset of Acute Rejection.

BACKGROUND: The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS: Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS: Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS: This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

Mixed Metal Oxide Nanoparticle Formulations for the Treatment of Seroma.

Seroma formation is a well-recognized postoperative complication for many plastic and general surgical procedures. Although various tissue adhesives and substances have been used in an effort to treat seroma formation, no therapies have been established clinically. Recently, the nano-bridging phenomenon has been introduced as a promising approach to achieve tissue adhesion and strong closure of deep skin wounds in rats. The present study seeks to assess the potential of nano-bridging beyond skin wounds in a rat model of seroma. Seromas were induced in 20 Lewis rats through bilateral axillary lymphadenectomy, excision of the latissimus dorsi and cutaneous maximus muscles, and disruption of dermal lymphatics. On postoperative day (POD) 7, the seroma was aspirated on both sides. A bioactive nanoparticle (NP) suspension based on zinc-doped strontium-substituted bioglass/ceria nanoparticles (NP group) or fibrin glue (fibrin group) was injected into the right seroma cavity, while the left side was left untreated. On POD 14, the NP group showed complete remission (no seromas at all), while the fibrin group recorded a reduction of only 63% in the seroma fluid volume. The NPs exerted local anti-inflammatory and neo-angiogenic effects, without any detectable systemic changes. Moreover, the ceria levels recorded in the organs did not surpass the background level, indicating that the nanoparticles stayed at the site of application. This study is a promising first example demonstrating the ability of inorganic nanoparticle formulations to reduce seroma formation in a rat model, without any detectable systemic adverse effects. These results emphasize the potential of nanotechnological solutions in the therapeutic management of seroma in the clinical setting.

Workhorse Free Functional Muscle Transfer Techniques for Smile Reanimation in Children with Congenital Facial Palsy: Case Report and Systematic Review of the Literature.

BACKGROUND: Pediatric facial palsy represents a rare multifactorial entity. Facial reanimation restores smiling, thus boosting self-confidence and social integration of the affected children. The purpose of this paper is to present a systematic review of microsurgical workhorse free functional muscle transfer procedures with emphasis on the long-term functional, aesthetic, and psychosocial outcomes. MATERIALS AND METHODS: We performed a literature search of the PubMed database from 1995 to 2019 using the following search strategy: "facial paralysis"[Title/Abstract] OR "facial palsy"[Title]. We used as limits: full text, English language, age younger than 18 years, and humans. Two independent reviewers performed the online screening process using Covidence. Forty articles met the inclusion criteria. The protocol was aligned with the PRISMA statement (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and was registered at the International Prospective Register of Systematic Reviews (PROSPERO, CRD42019150112) of the National Institute for Health Research. RESULTS: Free functional muscle transfer procedures include mainly segmental gracilis, latissimus dorsi, and pectoralis minor muscle transfer. Facial reanimation procedures with the use of the cross-face nerve graft (CFNG) or masseteric nerve result in almost symmetric smiles. The transplanted muscle grows harmoniously along with the craniofacial skeleton. Muscle function and aesthetic outcomes improve over time. All children presented improved self-esteem, oral commissure opening, facial animation, and speech. CONCLUSIONS: A two-stage CFNG plus an FFMT may restore a spontaneous emotive smile in pediatric facial palsy patients. Superior results of children FFMT compared to adults FFMT are probably attributed to greater brain plasticity.

Arterio-venous malformations of the ear: Description of distinct anatomical presentation and multidisciplinary management approach.

BACKGROUND: Arterio-venous malformations (AVMs) of the ear are a rare entity and their management should be decided in a dedicated multidisciplinary team (MDT) setting. The aim of this study is to describe the distinct anatomical patterns of the auricular AVMs in our unit and propose a combined interventional radiological and surgical approach. MATERIALS AND METHODS: All consecutive patients presenting with AVMs of the ear and reviewed by the Vascular Anomalies MDT between 2014 and 2019 were included in this study. Signs, symptoms, diagnostic investigations and operative findings were collected prospectively. RESULTS: After reviewing our nine patients, we identified four anatomical patterns of auricular AVMs: I: involves just a component of the ear and should undergo embolization followed by excision and reconstruction without significant loss of form; II: affects the superior two-thirds of the ear, sparing the lobule and part of the conchal bowl; these patients should undergo embolization, excision and monitoring before formal reconstruction of the ear is offered; III: involves the entire ear and should undergo embolization and pinnectomy; if there is no recurrence, the patients can be offered either a carved-rib cartilage reconstruction or a prosthesis, depending on the quality of the surrounding soft-tissues; IV: involves the ear and surrounding tissue, making surgical management and subsequent reconstruction extensive. CONCLUSION: The management of auricular AVMs is based on the extent of the ear involved. We feel that our combined interventional radiological and surgical approach will aid the management of these complex patients.

Predicting risk factors that lead to free flap failure and vascular compromise: A single unit experience with 565 free tissue transfers.

BACKGROUND: Even though the benefit of free tissue transfer is uncontested in complex reconstructive cases, vascular compromise and/or flap failure remain a challenge for the surgeon and identification of possible risk factors can aid in the preoperative planning. The aim of this study was to identify the individual risk factors leading to flap failure and/or vascular compromise in free tissue transfers in a single institution over a period of 10 years and to create an index predicting these problems, as well as finding predictors of other postoperative complications. METHODS: Data from all the patients undergoing free tissue transfers between 2009 and 2018 were retrospectively analyzed (demographics, comorbidities, flap failure, vascular compromise, and other complications). The results from the univariate and multivariate analyses were used to create an index. RESULTS: A predictability index with three classes (low, moderate, and high risk) was calculated for each patient, based on defect etiology and the presence of coronary heart disease, diabetes, smoking, peripheral arterial vascular disease, and arterial hypertension. A patient with moderate-risk index had 9.3 times higher chances of developing vascular compromise than those in the low-risk group, while a high-risk index had 18.6 higher odds (p=0.001). American Society of Anesthesiologists (ASA) classification was found to be a predictor of complications in free tissue transfer (p=0.001). CONCLUSION: If patients at a high risk of vascular compromise could be identified preoperatively through this predictability index, patient counseling could be improved and the surgeon might adapt the reconstructive plan and choose an alternative reconstructive strategy.

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.

Multiscale Analysis of Metal Oxide Nanoparticles in Tissue: Insights into Biodistribution and Biotransformation.

Metal oxide nanoparticles have emerged as exceptionally potent biomedical sensors and actuators due to their unique physicochemical features. Despite fascinating achievements, the current limited understanding of the molecular interplay between nanoparticles and the surrounding tissue remains a major obstacle in the rationalized development of nanomedicines, which is reflected in their poor clinical approval rate. This work reports on the nanoscopic characterization of inorganic nanoparticles in tissue by the example of complex metal oxide nanoparticle hybrids consisting of crystalline cerium oxide and the biodegradable ceramic bioglass. A validated analytical method based on semiquantitative X-ray fluorescence and inductively coupled plasma spectrometry is used to assess nanoparticle biodistribution following intravenous and topical application. Then, a correlative multiscale analytical cascade based on a combination of microscopy and spectroscopy techniques shows that the topically applied hybrid nanoparticles remain at the initial site and are preferentially taken up into macrophages, form apatite on their surface, and lead to increased accumulation of lipids in their surroundings. Taken together, this work displays how modern analytical techniques can be harnessed to gain unprecedented insights into the biodistribution and biotransformation of complex inorganic nanoparticles. Such nanoscopic characterization is imperative for the rationalized engineering of safe and efficacious nanoparticle-based systems.

Regional Lymphatic Inclusion in Orthotopic Hindlimb Transplantation: Establishment and Assessment of Feasibility in a Rodent Model.

BACKGROUND: The lymphatic system may play an important role in local immunomodulation in vascularized composite allotransplantation (VCA). Currently, there is no standardized VCA model that includes the regional draining lymphatic tissue. The aim of this study was to develop a rapid and efficient orthotopic hindlimb transplantation model in rats that included the draining lymphatic basin to permit further evaluation of the lymphatic system's role in VCA. METHODS: Thirty transplantations from Brown Norway rats to Lewis rats were performed. To include the regional lymphatic tissue, the superficial epigastric vessels were preserved to allow retrieval of the corresponding inguinal lymph nodes, including the inguinal fat pad, with the hindlimb. A cuff technique was used for the vein, whereas the conventional microsurgical technique was used for the arterial anastomosis. Vascular patency was confirmed through laser Doppler analysis at postoperative day 1 and histological analysis after euthanasia. RESULTS: The presence and vascularization of the inguinal lymph nodes were verified with indocyanine green lymphoscintigraphy at the time of transplantation. Mean total ischemia time was 69 ± 24 minutes, and mean recipient operation time was 80 ± 19 minutes. Overall transplant survival rate was 93.3%. Laser Doppler analysis showed vascular (technical) success, indocyanine green lymphoscintigraphy confirmed the presence of lymph nodes and the histological analysis revealed patent anastomoses. CONCLUSIONS: We successfully developed an experimental orthotopic hindlimb transplantation model in rats that includes the draining inguinal lymphatic basin, which is an important asset in further research on lymphatic tissue and its role in VCA.

The effects of optimizing blood inflow in the pedicle on perforator flap survival: A pilot study in a rat model.

BACKGROUND: Perforator flaps have led to a revolution in reconstructive surgery by reducing donor site morbidity. However, many surgeons have witnessed partial flap necrosis. Experimental methods to increase inflow have relied on adding a separate pedicle to the flap. The aim of our study was to experimentally determine whether increasing blood flow in the perforator pedicle itself could benefit flap survival. METHODS: In 30 male Lewis rats, an extended posterior thigh perforator flap was elevated and the pedicle was dissected to its origin from the femoral vessels. The rats were assigned to three groups: control (group I), acute inflow (group II) and arterial preconditioning (group III) depending on the timing of ligation of the femoral artery distal to the site of pedicle emergence. Digital planimetry was performed on postoperative day (POD) 7 and all flaps were monitored using laser Doppler flowmetry perioperatively and postoperatively in three regions (P1-proximal flap, P2-middle of the flap, P3-distal flap). RESULTS: Digital planimetry showed the highest area of survival in group II (78.12%±8.38%), followed by groups III and I. The laser Doppler results showed statistically significant higher values in group II on POD 7 for P2 and P3. At P3, only group II recorded an increase in the flow on POD 7 in comparison to POD 1. CONCLUSIONS: Optimization of arterial inflow, regardless if performed acutely or as preconditioning, led to increased flap survival in a rat perforator flap model.