NHS-POx-loaded patch versus fibrin sealant patch in a porcine robotic liver bleeding model.

BACKGROUND: The management of bleeding is paramount to any surgical procedure. With the increased use of less invasive laparoscopic and robotic methods, achieving hemostasis can be challenging since the surgeons cannot manually apply hemostatic agents directly onto bleeding tissue. In this study, we assessed the use of a pliable hemostatic sealant patch comprising fibrous gelatin carrier impregnated with poly(2-oxazoline) (NHS-POx) for hemostasis in robotic liver resection in a porcine bleeding model. METHODS: The NHS-POx-loaded patch (GATT-Patch), was first evaluated in a Feasibility Study to treat surgical bleeding in 10 lesions, followed by a Comparative Study in which the NHS-POx patch was compared to a standard-of-care fibrin sealant patch (TachoSil), in 36 lesions (superficial, resection, or deep injuries mimicking metastasectomies). For each lesion type, the NHS-POx and fibrin sealant patches were used in an alternating fashion with 18 lesions treated with NHS-POx and 18 with the fibrin patch. Animal preparation and surgical procedures were consistent across studies. The primary outcome was time to hemostasis (TTH) within 3 min for the Feasibility Study and within 5 min for the Comparative Study. RESULTS: In the Feasibility Study, 8 of the 10 NHS-POx-treated lesions achieved hemostasis at 30 s and 3 min. In the Comparative Study, all 18 NHS-POx patch-treated lesions and 9 of the 18 fibrin sealant patch-treated lesions achieved hemostasis at 5 min. Median TTH with NHS-POx vs fibrin sealant patch was 30 vs 300 s (P < 0.001). CONCLUSIONS: In this animal study, hemostasis during robotic liver surgery was achieved faster and more often with the NHS-POx loaded vs fibrin sealant patch.

A novel ex vivo perfusion-based mandibular pig model for dental product testing and training.

BACKGROUND: A translational ex vivo perfusion-based mandibular pig model was developed as an alternative to animal experiments, for initial assessment of biomaterials in dental and maxillofacial surgery and training. This study aimed to assess the face and content validity of the novel perfusion-based model. METHODS: Cadaveric porcine heads were connected to an organ assist perfusion device for blood circulation and tissue oxygenation. Dental professionals and dental trainees performed a surgical procedure on the mandibula resembling a submandibular extraoral incision to create bone defects. The bone defects were filled and covered with a commercial barrier membrane. All participants completed a questionnaire using a 5-point Likert scale to assess the face and content validity of the model. Validation data between the two groups of participants were compared with Mann-Whitney U test. RESULTS: Ten dental professionals and seven trainees evaluated the model for face and content validity. Participants reported model realism, with a mean face validity score of 3.9 ± 1.0 and a content validity of 4.1 ± 0.8. No significant differences were found for overall face and content validity between experts and trainees. CONCLUSION: We established face and content validity in a novel perfusion-based mandibular surgery model. This model can be used as an alternative for animal studies evaluating new biomaterials and related dental and maxillofacial surgical procedural training.

Proof-of-principle of a lung sealant based on functionalized polyoxazolines: experiments in an ovine acute aerostasis model.

OBJECTIVES: More effective lung sealants are needed to prevent prolonged pulmonary air leakage (AL). Polyoxazoline-impregnated gelatin patch (N-hydroxysuccinimide ester functionalized poly(2-oxazoline)s; NHS-POx) was promising for lung sealing ex vivo. The aim of this study is to confirm sealing effectiveness in an in vivo model of lung injury. METHODS: An acute aerostasis model was used in healthy adult female sheep, involving bilateral thoracotomy, amputation lesions (bronchioles Ø > 1.5 mm), sealant application, digital chest tube for monitoring AL, spontaneous ventilation, obduction and bursting pressure measurement. Two experiments were performed: (i) 3 sheep with 2 lesions per lung (N = 4 NHS-POx double-layer, N = 4 NHS-POx single-layer, N = 4 untreated) and (ii) 3 with 1 lesion per lung (N = 3 NHS-POx single-layer, N = 3 untreated). In pooled linear regression, AL was analysed per lung (N = 7 NHS-POx, N = 5 untreated) and bursting pressure per lesion (N = 11 NHS-POx, N = 7 untreated). RESULTS: Baseline AL was similar between groups (mean 1.38-1.47 l/min, P = 0.90). NHS-POx achieved sealing in 1 attempt in 8/11 (72.7%) and in 10/11 (90.9%) in >1 attempt. Application failures were only observed on triangular lesions requiring 3 folds around the lung. No influences of methodological variation between experiments was detected in linear regression (P > 0.9). AL over initial 3 h of drainage was significantly reduced for NHS-POx [median: 7 ml/min, length of interquartile range: 333 ml/min] versus untreated lesions (367 ml/min, length of interquartile range: 680 ml/min, P = 0.036). Bursting pressure was higher for NHS-POx (mean: 33, SD: 16 cmH2O) versus untreated lesions (mean: 19, SD: 15 cmH2O, P = 0.081). CONCLUSIONS: NHS-POx was effective for reducing early AL, and a trend was seen for improvement of bursting strength of the covered defect. Results were affected by application characteristics and lesion geometry.

Validity of a novel ex vivo porcine liver perfusion model for studying haemostatic products.

INTRODUCTION: We developed a novel normothermic ex vivo porcine liver perfusion model with whole blood in order to have alternatives for animal experiments in the research and development of new local haemostatic agents. This study aims to assess the construct and content validity of this model. METHODS: In this study we performed two ex vivo experiments using nine livers and one in vivo experiment using six female Norsvin Topigs pigs: (1) ex vivo liver perfusion for establishing physiological blood parameters of the perfused liver and controlled heparinization, (2) ex vivo liver perfusion with a surgical injury and (3) a surgical liver injury in anaesthetized pigs with and without heparin. RESULTS: Ex vivo coagulation parameters were comparable to in vivo with heparin. Blood gas values and metabolic parameters were comparable between ex vivo and in vivo with heparin, but significantly different compared with in vivo baseline, with the exception of (partial pressure of oxygen (PO2). Activated clotting time (ACT) values significantly differed depending on the heparin doses. The coagulation parameters fibrinogen, activated partial thromboplastin time, prothrombin time and ACT were rather constant during the 4 h ex vivo perfusion. Haemostatic efficacy of commercially available products was comparable between in vivo with heparin and the ex vivo liver perfusion experiment. CONCLUSION: This novel ex vivo liver perfusion model demonstrates good construct and content validity for at least 4 h of perfusion. The model is an easily accessible, table-top, tunable and effective alternative for the in vivo testing of (new) haemostatic products on their haemostatic properties.Validité d'un nouveau modèle de perfusion hépatique porcin ex-vivo pour l'étude des produits hémostatiques Résumé.

Efficacy of a novel polyoxazoline-based hemostatic patch in liver and spleen surgery.

BACKGROUND: A new hemostatic sealant based on a N-hydroxy-succinimide polyoxazoline (NHS-POx) polymer was evaluated to determine hemostatic efficacy and long-term wound healing and adverse effects in a large animal model of parenchymal organ surgical bleeds. METHODS: Experiment 1 included 20 pigs that were treated with two NHS-POx patch prototypes [a gelatin fibrous carrier (GFC) with NHS-POx and an oxidized regenerated cellulose (ORC) with poly(lactic-co-glycolic acid)-NHS-POx:NU-POx (nucleophilically activated polyoxazoline)], a blank gelatin patch (GFC Blank), TachoSil® and Veriset™ to stop moderate liver and spleen punch bleedings. After various survival periods (1-6 weeks), pigs were re-operated to evaluate patch degradation and parenchymal healing. During the re-operation, experiment 2 was performed: partial liver and spleen resections with severe bleeding, and hemostatic efficacy was evaluated under normal and heparinized conditions of the two previous prototypes and one additional NHS-POx patch. In the third experiment an improved NHS-POx patch (GATT-Patch; GFC-NHS-POx and added 20% as nucleophilically activated polyoxazoline; NU-POx) was compared with TachoSil®, Veriset™ and GFC Blank on punch bleedings and partial liver and spleen resections for rapid (10s) hemostatic efficacy. RESULTS: NHS-POx-based patches showed better (GFC-NHS-POx 83.1%, ORC-PLGA-NHS-POx: NU-POx 98.3%) hemostatic efficacy compared to TachoSil® (25.0%) and GFC Blank (43.3%), and comparable efficacy with Veriset™ (96.7%) on moderate standardized punch bleedings on liver and spleen. All patches demonstrated gradual degradation over 6 weeks with a reduced local inflammation rate and an improved wound healing. For severe bleedings under non-heparinized conditions, hemostasis was achieved in 100% for Veriset™, 40% for TachoSil and 80-100% for the three NHS-POx prototypes; similar differences between patches remained for heparinized conditions. In experiment 3, GATT-Patch, Veriset™, TachoSil and GFC Blank reached hemostasis after 10s in 100%, 42.8%, 7.1% and 14.3%, respectively, and at 3 min in 100%, 100%, 14.3% and 35.7%, respectively, on all liver and spleen punctures and resections. CONCLUSIONS: NHS-POx-based patches, and particularly the GATT-Patch, are fast in achieving effective hemostatic sealing on standardized moderate and severe bleedings without apparent long-term adverse events.

Sealing effectiveness of a novel NHS-POx based patch: experiments in a dynamic ex vivo porcine lung.

Background: Sealants are used to prevent prolonged pulmonary air leakage (PAL) after lung resections (incidence 5.6-30%). However, clinical evidence to support sealant use is insufficient, with an unmet need for a more effective product. We compared a novel gelatin patch impregnated with functionalized polyoxazolines (NHS-POx) (GATT-Patch) to commercially available sealant products. Methods: GATT-Patch Single/Double layers were compared to Progel®, Coseal®, Hemopatch® and TachoSil® in an ex vivo porcine lung model (first experiment). Based on these results, a second head-to-head comparison between GATT-Patch Single and Hemopatch® was performed. Air leakage (AL) was assessed in three settings using increasing ventilatory pressures (max =70 cmH2O): (I) baseline, (II) with 25 mm × 25 mm superficial pleural defect, and (III) after sealant application. Lungs floating on saline (37 ℃) were video recorded for visual AL assessment. Pressure and tidal volumes were collected from the ventilator, and bursting pressure (BP), AL and AL-reduction were determined. Results: Per sealant 10 measurements were performed (both experiments). In the first experiment, BP was superior for GATT-Patch Double (60±24 cmH2O) compared to TachoSil® (30±11 cmH2O, P<0.001), Hemopatch® (33±6 cmH2O, P=0.006), Coseal® (25±13 cmH2O, P=0.001) and Progel® (33±11 cmH2O, P=0.005). AL-reduction was superior for GATT-Patch Double (100%±1%) compared to Hemopatch® (46%±50%, P=0.010) and TachoSil® (31%±29%, P<0.001), and also for GATT-Patch Single (100%±14%, P=0.004) and Progel (89%±40%, P=0.027) compared to TachoSil®. In the second experiment, GATT-Patch Single was superior regarding BP (45±10 vs. 40±6 cmH2O, P=0.043) and AL-reduction (100%±11% vs. 68%±40%, P=0.043) when compared to Hemopatch®. Conclusions: The novel NHS-POx patch shows promise as a lung sealant, demonstrating elevated BP, good adhesive strength and a superior AL-reduction.

Intrinsic pulmonary sealing, its mechanisms and impact on validity and translational value of lung sealant studies: a pooled analysis of animal studies.

Background: No validated and standardized animal models of pulmonary air leakage (PAL) exist for testing aerostatic efficacy of lung sealants. Lack of negative control groups in published studies and intrinsic sealing mechanisms of healthy animal lungs might contribute to a translational gap, leading to poor clinical results. This study aims to address the impact of intrinsic sealing mechanisms on the validity of PAL models, and investigate the conditions required for an ovine model of PAL for lung sealant testing. Methods: An ovine acute aerostasis model was developed, consisting of a bilateral thoracotomy with lesion creation, chest tube insertion and monitoring of air leaks using digital drains (≥80 minutes), under spontaneous respiration. Healthy mixed-breed adult female sheep were used and all in vivo procedures were performed under terminal anesthesia. Superficial parenchymal lesions were tested post-mortem and in vivo, extended lesions including bronchioles (deep bowl-shaped and sequential lung amputation lesions) were tested in vivo. Experiment outcomes include air leakage (AL), minimal leaking pressure (MLP) and histology. Results: Two post-mortem (N=4 superficial parenchymal lesions) and 10 in vivo experiments (N=5 superficial parenchymal and N=16 lesions involving bronchioles) were performed. In contrast to the post-mortem model, superficial parenchymal lesions in vivo showed less air leak [mean flow ± standard deviation (SD): 760±693 vs. 42±33 mL/min, P=0.055]. All superficial parenchymal lesions in vivo sealed intrinsically within a median time of 20 minutes [interquartile range (IQR), 10-75 minutes]. Histology of the intrinsic sealing layer revealed an extended area of alveolar collapse below the incision with intra-alveolar hemorrhage. Compared to superficial parenchymal lesions in vivo, lesions involving bronchioles induced significantly higher air leak post-operatively (normalized mean flow ± SD: 459±221 mL/min, P=0.003). At termination, 5/9 (55.6%) were still leaking (median drain time: 273 minutes, IQR, 207-435 minutes), and intrinsic sealing for the remaining lungs occurred within a median of 115 minutes (IQR, 52-245 minutes). Conclusions: Lung parenchyma of healthy sheep shows a strong intrinsic sealing mechanism, explained pathologically by an extended area of alveolar collapse, which may contribute to a translational gap in lung sealant research. A meaningful ovine model has to consist of deep lesions involving bronchioles of >⌀1.5 mm. Further research is needed to develop a standardized PAL model, to improve clinical effectiveness of lung sealants.

New polyoxazoline loaded patches for hemostasis in experimental liver resection.

A new cost-effective NHS functionalized polyoxazoline (POx) loaded polymer with strong hemostatic properties has been developed. In this study, we investigate POx loaded hemostatic patches regarding hemostatic efficacy, local inflammatory reaction and wound-healing, as compared to the non-POx treated blanks and commercially available hemostatic products. Hundred and ten rats divided into 11 groups of 10 animals underwent partial liver lobe resection. Eight groups received experimental patches, two groups commercially available hemostatic patches (TachoSil® and Veriset™, positive controls), one group with gauzes (negative control). Each animal received twice a patch with a size 1.5 × 2.5 cm, on each partially resected lobe. Primary endpoint was time to hemostasis (TTH). The rats were sacrificed at different time points (1, 3, or 7 days) to measure local inflammatory response and early wound healing. Of the POx loaded patches, GFC NHS-POx (TTH 20.4 s, p = .019) and GFC-NHS-POx1.5 (TTH 0.0 s, p = .003) showed significantly faster TTH compared to TachoSil® (TTH 95.4 s), and were comparable to Veriset™ (TTH 17.0 s). Three patches, GFC-NHS-POx 1.5 (TTH 0.0 s, p = .016), ORC NHS-POx:NU-POx (TTH 91.4 s, p = .033), and ORC-PLGA NHS-POx:NU-POx (TTH 105.6 s, p = .04) had a lower TTH compared to their own blank carrier (TTH 74.9, 157.8, and 195.7 s, respectively). With regard to biocompatibility, all POx loaded patches showed results comparable to TachoSil® and Veriset™. NHS-POx-loaded hemostatic patch demonstrate fast and effective hemostasis, comparable or better than commercially available hemostatic patches, with similar early biocompatibility.

Tubular collagen scaffolds with radial elasticity for hollow organ regeneration.

Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs. STATEMENT OF SIGNIFICANCE: In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs.