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Since its clinical implementation, microvascular surgery has depended on the continuous improvement of magnification tools. One of the more recent developments is a high-definition three-dimensional (3D) digital system (exoscope), which provides an alternative to the state-of-the-art operating microscopes. This study aimed to evaluate the advantages and disadvantages of this technology and compare it with its predecessor. The study included 14 surgeons with varying levels of experience, none of which had used a 3D optical system previously. Six of these surgeons performed five arterial and five venous anastomoses in the chicken thigh model with both the VITOM 3D exoscope-guided system and the Pentero operating microscope. These anastomoses were then evaluated for their quality and anastomosis time. The participants and the other eight surgeons, who had used the digital 3D camera system for microsurgical training exercises and vascular sutures, answered a questionnaire. The anastomosis time and number of complications were lower with the conventional microscope. Participants rated the image quality with the conventional microscope as higher, whereas the field of view and ergonomics were favorable in the digital 3D camera system. Exoscopes are optics suitable for performing simple microvascular procedures and are superior to classical microscopes ergonomically. Thus far, they are inferior to classical microscopes in terms of image quality and 3D imaging.
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Microscopia , Microcirurgia , Microcirurgia/métodos , Procedimentos Neurocirúrgicos/métodos , Anastomose Cirúrgica/métodos , Artérias , Imageamento Tridimensional/métodosRESUMO
BACKGROUND: Perforator imaging is a prerequisite in preoperative planning of the peroneal perforator flap and the fibula skin island. Although reports indicate that indocyanine green angiography assessment method might be advantageous over conventional ultrasound-based techniques (i.e., Doppler and color duplex), in practice, clear evidence is lacking. Thus, a comparative assessment of the utility of indocyanine green angiography and ultrasound-based techniques in the identification of suitable lower leg skin perforators was performed. METHODS: A prospective clinical cohort study with a series of 12 consecutive patients was conducted to assess indocyanine green angiography, Doppler ultrasound, and color duplex ultrasound techniques for preoperative perforator detection in the lower leg before free fibula flap harvest. Anatomical dissection served as a reference. Parameters measured were perforator spatial distance to the reference (precision), operative time expenditure, and ease of device usage for assessment/outcomes. RESULTS: This study included 12 patients, with a total of 27 perforators. Exhibition of technique sensitivity and positive predictive values were as follows: indocyanine green angiography, 93 percent and 100 percent; Doppler ultrasound, 82 percent and 82 percent; and color duplex ultrasound, 89 percent and 86 percent, respectively. With regard to the indocyanine green angiography technique, the distance to the actual perforator location was significantly shorter, which aided detection and lesser time expenditure during operation. CONCLUSIONS: The indocyanine green angiography technique proved to have high precision, sensitivity, positive predictive value, and easy-to-use capabilities because of its exceptional spatial and temporal information, compared to the conventional, ultrasound-based techniques. Therefore, indocyanine green angiography is superior for preoperative perforator imaging of the lateral lower leg. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, II.
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Verde de Indocianina , Retalho Perfurante , Angiografia/métodos , Estudos de Coortes , Humanos , Retalho Perfurante/irrigação sanguínea , Estudos Prospectivos , Artérias da TíbiaRESUMO
Systematic assessment of computed tomography (CT) scans and clinical symptoms is necessary to quickly indicate the correct treatment of zygomatico-orbital (ZMO) fractures. For this purpose, a clinical scoring system (=Clinical Score) was developed and correlated with CT scans to analyse its validity. Every operated, isolated, and unilateral ZMO fracture between January 2012 and December 2016 was screened retrospectively, including patient and treatment data. All available CT scans were analysed, and the grade of dislocation was measured for each case and plane. Four hundred and sixty-one cases were included and showed a median surgery time of 66.0 min (5.0−361.0) and a median postoperative hospital stay of three days (0−25). The distribution of gender, aetiologies and age groups was significantly different (each p = 0.001), and the aetiology had a significant influence on the Clinical Score (p = 0.038). The degree of dislocation in the coronary and sagittal planes correlated significantly with the Clinical Score with regard to the orbital involvement (p < 0.001, ρ = 0.566; p < 0.001, ρ = 0.609). The simple, quick, and easy-to-apply Clinical Score showed a significant correlation with the most important planes in CT scans as well as with the clinical course. It may facilitate fast risk stratification of the patient. However, the validity of the proposed score in determining indications must now be evaluated in a prospective setting, including both operated and non-operated fractures.
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BACKGROUND: The vessel-depleted, irradiated, and frozen neck, as well as severe atherosclerosis of recipient vessels represent challenging problems in free flap transfer. Extracorporeal free flap perfusion theoretically allows free flap reconstructions in the absence of local donor vessels, but is associated with a number of technical issues. In this study, a novel technique is presented using a commercially available system for extracorporeal membrane oxygenation (ECMO), modified for small blood volumes. METHODS: After preclinical testing, an ECMO system certified for lung support was used to establish blood flow through the flap's artery with oxygenation, decarboxylation and warming of diluted packed blood cells. Venous blood was allowed to flow passively into a separate container. Perfusion was performed for 15 min at intervals of 4 h over 4-6 days. RESULTS: Five patients with soft tissue defects requiring free flap reconstruction were included. Either primarily thinned anterolateral thigh (ALT) flaps (n = 3) or radial forearm flaps (n = 2) were used. We observed infection of the perfusate, with consequent subtotal flap loss, in one patient, complete epithelial loss in two patients, venous congestion in one case, and almost uneventful healing in the fifth patient. With conservative wound care and a split thickness skin graft in one case, stable wound coverage was achieved in all patients except one, who had secondary healing. None of the patients required a second flap for sufficient coverage. CONCLUSIONS: The technique described is associated with the risks of infection, flap congestion, nutritive hypoperfusion, and consequent tissue loss. Nevertheless, stable defect closure seems to be achievable even in patients with depleted recipient vessels.
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Oxigenação por Membrana Extracorpórea , Retalhos de Tecido Biológico , Procedimentos de Cirurgia Plástica , Humanos , Transplante de Pele , Coxa da PernaRESUMO
Allogenic graft material and tissue engineering have recently shown promising results for the improvement of both esthetic and functional outcomes in the treatment of large skin defects. We chose human amniotic membrane as a cellular scaffold in order to develop a skin substitute for later in vivo uses. Various methods of de-epithelialization of the human amniotic membrane were evaluated by histological analysis including hematoxylin-eosin and laminin staining, optic coherence tomography, and scanning electron microscopy with 0.25/0.02% trypsin/ethylenediaminetetraacetic acid treatment and mechanical cell removal showing an almost complete loss of the epithelium and a mainly intact basement membrane. Novel examination of human amniotic membrane by optic coherence tomography was feasible, but difficulties were experienced in handling and interpretation of the tissue as no comparable data exist. Subsequently, we developed an air-liquid interface cell culture to cultivate keratinocytes and fibroblasts on the de-epithelialized human amniotic membrane. We achieved a mostly keratinized surface on the epidermal side with a confluent fibroblast network on the chorion side.
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Autologous cartilage as donor tissue for various surgical reconstructions such as nasal septum regeneration is limited and associated with donor site morbidity. Our goal was to evaluate a new resorbable chondroconductive biomaterial made of decellularized porcine nasal septum cartilage compared with autologous native auricular cartilage as the gold standard. In order to examine the material and determine its long-term outcome further, we used subcutaneous implantation and septal implantation in an orthotopic rabbit model. In addition to non-seeded decellularized xenogenic cartilage, chondrocyte-seeded decellularized xenogenic cartilage was implanted as a septal replacement. After a three- or six-month period, the formation of newly synthesized cartilage extracellular matrix was evaluated immunohistochemically, whereas septal integrity and biocompatibility were evaluated histologically. The formation of the implanted neoseptum and form stability was analyzed by using 7-Tesla Magnetic Resonance Imaging. Good biocompatibility with no excessive rejection was demonstrated in all groups. Long-term stable and reliable septal reconstruction could be achieved in the study groups with or without cell seeding with autologous auricular chondrocytes. Autologous cell seeding was advantageous only with regard to septal perforations. Thus, cell seeding provides a benefit regarding long-term stability. However, because of slightly better biocompatibility, less pronounced septum deviation and the markedly lower effort involved, the non-seeded scaffold is favoured for possible clinical application.
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Condrócitos , Engenharia Tecidual , Animais , Matriz Extracelular , Cartilagens Nasais , Coelhos , Regeneração , Suínos , Alicerces TeciduaisRESUMO
Human amniotic membranes (hAMs) have shown promising results in recent studies aimed at improving wound healing through several mechanisms. We wanted to investigate its properties as a scaffold by adding autologous cells to treat full-thickness skin defects and hypothesized that recultivated hAM would show an even improved wound healing by accelerating the epidermal closure of the wound. METHODS: In an air-liquid cell culture, we cultivated autologous keratinocytes and fibroblasts on the hAM until a mostly keratinized surface was achieved. These hAM, de-epithelialized hAM, native hAM with remaining allogenous cells, and negative controls were compared in the treatment of circular 30 × 30 mm2 full-thickness skin defects in 4 groups of 6 rats with one wound each. We evaluated the wound contraction every 10 days until wound closure, the macroscopic scar appearance on the Vancouver Scar Scale and the qualitative histological properties of the scar regarding morphology and continuity of the basement membrane. RESULTS: Rats treated with de-epithelialized hAM showed more extent wound contraction (P < 0.001) than the other 3 groups, which did not differ significantly compared with the control group (P > 0.05). Vancouver Scar Scale showed no significantly statistical differences between the 4 groups (P = 0.46). The scar structure of all rats showed similar morphologies, the only difference being the absence of a basement membrane in the negative controls compared with the groups treated with hAM. CONCLUSION: The rats treated with hAM showed no improved wound healing but a tendency toward a more prominent basement membrane in the resulting scar.
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A critical limitation in the cultivation of cartilage for tissue engineering is the dedifferentiation in chondrocytes, mainly during in vitro amplification. Despite many previous studies investigating the influence of various conditions, no data exist concerning the effects of hypothermia. Our aim has been to influence chondrocyte dedifferentiation in vitro by hypothermic conditions. Chondrocytes were isolated from cartilage biopsies and seeded in monolayer and in three-dimensional pellet-cultures. Each cell culture was either performed at 32.2°C or 37°C during amplification. Additionally, the influence of the redifferentiation of chondrocytes in three-dimensional cell culture was examined at 32.2°C and 37°C after amplification at 32.2°C or 37°C. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure cell proliferation in monolayer, whereas the polymerase chain reaction and immunohistochemical and histological staining were used in three-dimensional pellet-cultures. Real-time polymerase chain reaction was employed to measure the relative expression of the target genes collagen II, collagen I, aggrecan and versican. Ratios were estimated between collagen II/collagen I and aggrecan/versican to evaluate differentiation. A higher value of these ratios indicated an advantageous status of differentiation. In monolayer, hypothermia at 32.2°C slowed down the proliferation rate of chondrocytes significantly, being up to two times lower at 32.2°C compared with culture at 37°C. Simultaneously, hypothermia in monolayer decelerated dedifferentiation. The ratio of aggrecan/versican was significantly higher at 32.2°C compared with that at 37°C. In three-dimensional pellet-culture, the chondrocytes redifferentiated at 32.2°C and at 37°C, and this process is more distinct at 37°C than at 32.2°C. Similar results were obtained for the ratios of collagen II/collagen I and aggrecan/versican and were supported by immunochemical and histological staining. Thus, hypothermic conditions for chondrocytes are mainly advantageous in monolayer culture. In three-dimensional pellet-culture, redifferentiation predominates at 37°C compared with at 32.2°C. In particular, the results from the monolayer cultures show potential in the avoidance of dedifferentiation.
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Human adipose-derived stem cells (ASC) have been shown to differentiate into mature adipocytes and to play an important role in creating the vasculature, necessary for white adipose tissue to function. To study the stimulatory capacity of ASC on endothelial progenitor cells we used a commercially available co-culture system (V2a - assay). ASC, isolated from lipoaspirates of 18 healthy patients, were co-cultured for 13 d on endothelial progenitor cells. Using anti CD31 immunostaining, cells that had undergone endothelial differentiation were quantified after the defined co-cultivation period. Endothelial cell differentiation was observed and demonstrated by an increase in area covered by CD31+ cells compared with less to no endothelial cell differentiation in negative and media-only controls. Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) in supernatant medium collected during the co-cultivation period revealed elevated VEGF levels in the co-culture samples as compared with ASC cultures alone, whereas no increase in adiponectin was detected by ELISA. These findings help to provide further insights in the complex interplay of adipose derived cells and endothelial cells and to better understand the diversity of ASCs in respect of their stimulatory capacity to promote angiogenesis in vitro.
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Adipócitos/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/citologia , Idoso , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVES/HYPOTHESIS: Septal deviation and hypertrophic inferior turbinates are a frequent cause of nasal breathing disorders. The goal of this study was to prove the effectiveness and safety of three current turbinoplasty techniques. STUDY DESIGN: This is a prospective, three-arm, single-blinded, single-center, randomized controlled trial. METHODS: Sixty patients were randomly assigned to either anterior turbinoplasty (ATP) (n = 20), radiofrequency ablation (RFA) (n = 19; Celon Pro Breath), or novel submucous radial diode laser ablation (DLA) (n = 21; ELVeS Radial PainLess, wavelength = 1,470 nm), each in combination with standard septoplasty. Acoustic rhinometry, rhinomanometry, subjective nose questionnaire, and saccharin test served as outcome parameters for preoperative and 3-month, 1-year, and 2-year postoperative examinations. RESULTS: After 3 months 47/60 patients were evaluated, 28/60 patients were evaluated after 1 year, and 26/60 patients were evaluated in the 2-year follow-up visit. An improvement of nasal breathing was observed in all three groups in all follow-up visits. The increase of endonasal volume 2 (volume between the nasal valve and body of the inferior turbinate) was statistically significant in the ATP and RFA group after 3 months and 2 years, and in the RFA group also after 1 year. The DLA group failed to reach significance level in all follow-up visits. Subjective evaluation of nasal breathing improved in all three groups. CONCLUSIONS: In this trial, three different current techniques of turbinate surgery in combination with standard septoplasty were effective for the improvement of nasal breathing. The ATP and RFA techniques were more effective in the long term than DLA. LEVEL OF EVIDENCE: 1b. Laryngoscope, 2016 127:303-308, 2017.
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Obstrução Nasal/cirurgia , Septo Nasal/anormalidades , Septo Nasal/cirurgia , Conchas Nasais/cirurgia , Adulto , Ablação por Cateter , Terapia Combinada , Feminino , Seguimentos , Humanos , Hipertrofia , Terapia a Laser , Lasers Semicondutores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Obstrução Nasal/etiologia , Obstrução Nasal/patologia , Estudos Prospectivos , Método Simples-Cego , Conchas Nasais/patologia , Adulto JovemRESUMO
The warm ischaemia time of microvascular free flaps is limited. Incalculable events, such as lack of adequate recipient vessels or intraoperative medical emergencies, can lead to prolonged ischaemia and potentially to flap loss. In this study, critically perfused ischaemic or congested flaps were temporarily perfused with an extracorporeal perfusion system until anastomosis could be commenced. Temporary extracorporeal perfusion was performed in 8 radial forearm flaps for 147 ± 52 (range 77-237) minutes. Flap perfusion was assessed using Indocyanine Green fluorescence angiography and combined laser Doppler flowmetry and remission spectroscopy. Results were compared with those of 30 patients who underwent conventional reconstruction with radial forearm flaps. Flap survival, flap microcirculation, postoperative complications, and hospital stay did not differ between groups. We report successful free flap transfer after short-term extracorporeal perfusion for up to 4 h in 8 patient cases. Temporary extracorporeal free flap perfusion reduces the warm ischaemia time in emergency situations and can help to prevent flap failure in critically perfused or congested flaps. The trial is registered with ClinicalTrials.gov, number NCT02449525.
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Oxigenação por Membrana Extracorpórea/métodos , Retalhos de Tecido Biológico/irrigação sanguínea , Adulto , Idoso , Carcinoma de Células Escamosas/cirurgia , Oxigenação por Membrana Extracorpórea/instrumentação , Feminino , Angiofluoresceinografia , Antebraço/cirurgia , Retalhos de Tecido Biológico/cirurgia , Humanos , Isquemia/terapia , Masculino , Reconstrução Mandibular/métodos , Microcirculação , Pessoa de Meia-Idade , Neoplasias Bucais/cirurgiaRESUMO
Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco's modified Eagle medium Ham's F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).
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Criopreservação/métodos , Crioprotetores/farmacologia , Células Endoteliais/citologia , Congelamento , Vitrificação , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Camundongos , Vitrificação/efeitos dos fármacosRESUMO
BACKGROUND: Free flap transplantation may not be feasible in patients with inadequate or absent recipient vessels. We report successful mandibular composite reconstructions without anastomosis in three consecutive patients with vessel-depleted neck. Based on clinical reports describing early neovascularisation, temporary extracorporeal perfusion of flaps was maintained until the flaps had become independent from the extracorporeal blood supply. METHODS: A blood transfusion bag filled with the patients' arterialised blood was connected to the flap artery and set under rhythmic compression to ensure continuous blood supply to the flap. The returning venous blood was collected but not reinfused. Extracorporeal circulation was sustained for 10-13 days until flaps had become independent from the external blood supply. Flap viability was assessed every 2 h using combined laser Doppler flowmetry and remission spectroscopy. RESULTS: Successful bony reconstructions were achieved in all three consecutive patients substantiated by MRI-, CT-scan or bone scintigraphy. Neovascularisation occurred within the soft tissues of all flaps with the exception of one skin paddle, which later developed necrosis. Systemic transfusion of 12-25 units of packed red cells was necessary to compensate for the blood loss. CONCLUSIONS: With this technique, transplantation of composite free flaps becomes feasible even in the absence of recipient vessels, opening up new treatment options to a broad range of complex surgical problems. Blood reinfusion should be pursued in the future to avoid excessive blood transfusions. The trial is registered with ClinicalTrials.gov, number NCT02449525.
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Oxigenação por Membrana Extracorpórea/métodos , Retalhos de Tecido Biológico/irrigação sanguínea , Retalhos de Tecido Biológico/transplante , Reconstrução Mandibular/métodos , Artérias , Oxigenação por Membrana Extracorpórea/instrumentação , Retalhos de Tecido Biológico/cirurgia , Humanos , Fluxometria por Laser-Doppler , Microcirculação , Pescoço , Procedimentos de Cirurgia Plástica/métodos , Retalhos CirúrgicosRESUMO
BACKGROUND: Ketamine-xylazine is a frequently used combination for anesthesia in microsurgically operated rats and can be administered by intraperitoneal (IP) or intravenous (IV) injection. Both methods are associated with relatively high misadministration rates. In the present study, we want to introduce a femoral vein access that requires microsurgical cannulation but enables a 100% administration rate. METHODS: In this study, the maximal time of anesthesia was analyzed, time to response, latency time, and the total need for anesthetic agents in IP (n = 200) and IV (n = 40) anesthesia in Wistar rats for the purpose of microvascular operations. IV injections were achieved with an inserted microcatheter that was applied via a microsurgical femoral vein access. RESULTS: The time needed for the placement of the microcatheter was 5.76 ± 0.89 minutes. Maximal time of anesthesia (IP vs. IV) was 7.02 ± 1.92 versus 21.78 ± 5.77 hours (p < 0.0001), time to response was 137.5 ± 3.91 versus 18 ± 2.18 seconds (p < 0.0001), latency time 35.53 ± 3.21 versus 27.8 ± 2.88 minutes (p < 0.0001), and total volume of anesthetic 1.42 ± 0.39 versus 2.78 ± 0.73 mL (p < 0.0001), respectively. CONCLUSION: IV administration using the microsurgical femoral vein access is a feasible method with a quicker response rate and a 100% administration rate. Furthermore, it enables longer anesthesia, for example, complex microsurgical or other experimental procedures in the rat.
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Analgésicos/administração & dosagem , Anestésicos Dissociativos/administração & dosagem , Ketamina/administração & dosagem , Xilazina/administração & dosagem , Animais , Veia Femoral , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Masculino , Microcirurgia , Ratos WistarRESUMO
Bacterial nanocellulose (BNC), synthesized by the bacterium Gluconacetobacter xylinus, is composed of highly hydrated fibrils (99 % water) with high mechanical strength. These exceptional material properties make BNC a novel biomaterial for many potential medical and tissue engineering applications. Recently, BNC with cellulose content of 15 % has been proposed as an implant material for auricular cartilage replacement, since it matches the mechanical requirements of human auricular cartilage. This study investigates the biocompatibility of BNC with increased cellulose content (17 %) to evaluate its response in vitro and in vivo. Cylindrical BNC structures (Ø48 × 20 mm) were produced, purified in a built-in house perfusion system, and compressed to increase the cellulose content in BNC hydrogels. The reduction of endotoxicity of the material was quantified by bacterial endotoxin analysis throughout the purification process. Afterward, the biocompatibility of the purified BNC hydrogels with cellulose content of 17 % was assessed in vitro and in vivo, according to standards set forth in ISO 10993. The endotoxin content in non-purified BNC (2,390 endotoxin units (EU)/ml) was reduced to 0.10 EU/ml after the purification process, level well below the endotoxin threshold set for medical devices. Furthermore, the biocompatibility tests demonstrated that densified BNC hydrogels are non-cytotoxic and cause a minimal foreign body response. In support with our previous findings, this study concludes that BNC with increased cellulose content of 17 % is a promising non-resorbable biomaterial for auricular cartilage tissue engineering, due to its similarity with auricular cartilage in terms of mechanical strength and host tissue response.
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Celulose/administração & dosagem , Cartilagem da Orelha/fisiologia , Regeneração Tecidual Guiada/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Teste de Materiais , Animais , Celulose/metabolismo , Endotoxinas/análise , Gluconacetobacter xylinus/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , CoelhosRESUMO
The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15 × 8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.
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Pavilhão Auricular/irrigação sanguínea , Cartilagem da Orelha/irrigação sanguínea , Poliésteres/farmacologia , Retalhos Cirúrgicos/irrigação sanguínea , Alicerces Teciduais , Animais , Adesão Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/fisiologia , Pavilhão Auricular/citologia , Pavilhão Auricular/fisiologia , Pavilhão Auricular/cirurgia , Cartilagem da Orelha/citologia , Cartilagem da Orelha/fisiologia , Cartilagem da Orelha/cirurgia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Microcirurgia/métodos , Neovascularização Fisiológica , Poliésteres/química , Poliuretanos/química , Coelhos , Retalhos Cirúrgicos/transplante , Engenharia Tecidual/métodosRESUMO
Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.