Novel regenerative therapy combined with transphrenic peritoneoscopy-assisted omentopexy

Novel regenerative therapy combined with transphrenic peritoneoscopy-assisted omentopexy Abstract OBJECTIVES We previously reported that cell sheet transplantation combined with an omentopexy (OP) procedure is more effective for repairing heart damage when compared with cell sheet transplantation alone. However, a simultaneous (conventional) laparotomy as part of the OP may adversely affect the general condition of critically ill heart failure patients who would otherwise benefit from cell sheet transplantation, which is a paradox to be reconciled before this treatment can be applied in a clinical setting. We devised a novel endoscopic approach termed ‘transphrenic peritoneoscopy’ (TPP) for minimal access to abdominal organs from the thoracic cavity. Herein, we evaluated the feasibility and safety of TPP with an OP in a porcine myocardial infarction model. METHODS Myocardial infarction was induced in 4 mini pigs by placing an ameroid constrictor around the left anterior descending artery. One month later, a left thoracotomy was performed in 2 randomly selected mini pigs, and a laparoscopic port was placed on the left diaphragm to gain access into the abdominal cavity. Using a low-pressure pneumoperitoneum, a flexible gastrointestinal endoscope was advanced, then the omentum was partially grasped with endoscopic forceps and brought back into the thoracic cavity via the diaphragm. Skeletal myoblast cell sheets were then implanted over the impaired myocardium, followed by placing the omentum over the sheets. RESULTS TPP-assisted OP was accomplished in 2 post-myocardial infarction mini pigs with severe heart failure with an intra-abdominal pressure ≤8 mmHg within 30 min (22 and 27 min, respectively). Necropsy findings revealed a viable omentum flap and pedicle in both animals, with no evidence of procedure-related complications. Angiographic and histological analyses confirmed vessel communication between the omentum and the left ventricle. CONCLUSIONS Our TPP approach was shown to be feasible and safe with a low-pressure pneumoperitoneum, while the omentum flap was durable. This successful combination of techniques may provide less-invasive endoscopic intervention and regenerative therapy. Cell therapy, Laparotomy, Omentopexy, Omentum INTRODUCTION The omentum has historically been used for surgical revascularization in patients with ischaemic heart disease and is also known to release a number of angiogenic cytokines and attenuate inflammation [1, 2]. In addition, the gastroepiploic artery involved in the omentum flap plays an important role as an extracardiac blood source with high perfusion capacity for developing effective collateral vessels in the treatment for advanced coronary artery disease [3]. Because of its unique characteristics, an omentopexy (OP) procedure (transposition into the thoracic cavity through the diaphragm) has been clinically applied in the field of thoracic surgery to control infections, such as postoperative mediastinitis and empyema, as well as for cases of abdominal surgery to repair and prevent infections due to gastrointestinal anastomotic leakage and/or perforation [1, 4]. On the basis of this background, we have recently applied the omentum flap to the field of regeneration therapy and successfully established a unique therapeutic method that utilizes cell sheet transplantation combined with an OP in both small and large heart failure animal models [5–7]. Recently, we reported that this cell sheet transplantation method in combination with an OP was more effective for repairing a damaged heart when compared with cell sheet transplantation alone. However, a simultaneous (conventional) laparotomy as part of an OP may adversely affect the general condition of critically ill heart failure patients who would otherwise benefit from cell sheet transplantation through a left thoracotomy, which is a contradiction to be reconciled before this combined treatment can be applied in clinical settings. Although a laparoscopy-assisted approach for abdominal organs may be an alternative to a conventional laparotomy, the possibility that a pneumoperitoneum under usual intra-abdominal pressure (12–15 mmHg) causes a considerable decrease in venous return cannot be ignored, as it subsequently may lead to low cardiac output syndrome in patients with refractory heart failure [8]. To overcome the procedure-related issues noted earlier, we have devised a novel approach termed ‘transphrenic peritoneoscopy’ (TPP) for minimal access to abdominal organs from the thoracic cavity via a port placed on the diaphragm with a flexible gastrointestinal endoscope. This procedure is derived from the concept of ‘natural orifice transluminal endoscopic surgery’, which is a surgical technique whereby ‘scarless’ abdominal operations can be performed by inserting the endoscope through a natural orifice (e.g. mouth, urethra and anus) and entering the peritoneal cavity by making an incision on the luminal wall (e.g. stomach, vagina, bladder and colon) [9–11]. Herein, we evaluated the feasibility and safety of TPP-assisted OP in a porcine myocardial infarction (MI) model. In addition, we assessed its potential effectiveness for enhancing cell-sheet-mediated regenerative medicine. To the best of our knowledge, this is the first preclinical report on TPP, or transphrenic natural orifice transluminal endoscopic surgery, presented in the surgical literature. MATERIALS AND METHODS All experimental procedures were approved by our institutional ethics committee. Animal care was performed humanely in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Animal Resources and published by the National Institutes of Health (publication number 85-23, revised 1996). Study protocol MI was induced in 4 female mini pigs (8–10-month old, Kagoshima Miniature Swine Research Center, Kagoshima, Japan) each weighing 20–25 kg. Four weeks after MI, the animals were randomly divided into either the treatment (cell sheet transplantation plus TPP-assisted OP) or the control (non-treatment) group (n = 2 each). To assess the effect of treatment on LV volumes and function, electrocardiogram (ECG)-gated cardiac magnetic resonance imaging (MRI) was performed approximately 1 month after MI (e.g. 1 day prior to treatment) and 1 month after treatment with an 8-channel cardiac coil wrapped around the chest wall [12]. At the end-point of this study (1 month after treatment), the animals were humanely euthanized for histological and angiographical analyses of heart and omentum tissues. Creation of autologous skeletal muscle cell sheets Autologous skeletal muscle tissue weighing approximately 10–15 g was removed from the quadriceps femoris muscle, and then purified autologous skeletal muscle cells were cultured for 3 weeks to prepare for implantation, as previously described [6, 13, 14]. Briefly, the cells were incubated in 60-mm temperature-responsive culture dishes (UpCell®; Cellseed, Tokyo, Japan) at 37°C in 5% CO2 atmosphere for 24 h with the cell numbers adjusted to 1.5 × 107 cells per dish. The dishes were then transferred to another incubator set at 20°C for 1 h to release the cultured cells as an intact cell sheet. Using this protocol, skeletal muscle cells were spontaneously detached from the plate as a free-floating monolayer cell sheet. Generation of porcine chronic heart failure model Each mini pig was anaesthetized with an intravenous administration of ketamine hydrochloride (10 mg/kg; DAIICHI SANKYO, Tokyo, Japan) and xylazine (2 mg/kg; Bayer HealthCare, Leverkusen, Germany) for endotracheal intubation, and then anaesthesia was maintained with inhaled sevoflurane (2%). The pericardial space was exposed by a left thoracotomy through the 4th intercostal space. The distal portion of the left anterior descending coronary artery was directly ligated as ischaemic preconditioning to reduce the occurrence of lethal ventricular arrhythmia, followed by placement of an ameroid constrictor (COR-2.50-SS; Research Instruments SW, Escondido, CA, USA) around the left anterior descending coronary artery just distal to the left circumflex coronary artery branching [6, 13]. The muscle and skin were closed in layers, then anaesthesia was completed, and the animal was allowed to recover alone in a temperature-controlled cage. This technique produced an ischaemic cardiomyopathy model that reflected clinical relevance and was considered appropriate for preclinical studies with minimal procedure-related mortality. Cell sheet transplantation combined with transphrenic peritoneoscopy-assisted omentopexy Under general anaesthesia and double-lumen endotracheal intubation, a 12-mm standard laparoscopic port was created at the umbilical site with the animal in the supine position, and a standard laparoscopic CO2 insufflator (UHI-3, Olympus Corporation, Tokyo, Japan) was used to initiate and maintain the pneumoperitoneum with a pressure of 4 mmHg to check for any obvious abnormality in the abdominal cavity. After moving to a right lateral decubitus position, a second left thoracotomy was made under 1-lung ventilation, and the pericardium was incised to ensure a wider visual field of the left ventricle. The optimal delivery route of the pedicle omentum was determined by viewing directly from the left thoracotomy and intra-abdominal cavity through a rigid laparoscope. A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was then placed on the fixed site of the diaphragm to access the abdominal cavity, while paying close attention to not injure abdominal organs (Fig. 1A). A flexible gastrointestinal endoscope (GIF-Q260J, Olympus Medical Systems, Tokyo, Japan) was advanced into the intra-abdominal cavity through the port under the pneumoperitoneum with a pressure of 4 mmHg (up to 8 mmHg maximum if required) (Fig. 1B and C). The omentum was then identified and grasped with a reusable biopsy forceps (FG-47l-1, Olympus, Tokyo, Japan) and brought back to the pleural cavity via the diaphragm where the port was placed under 1-lung ventilation (Fig. 1D). The omentum grasped by the forceps was further mobilized with sufficient volume to cover the left ventricle, during which we confirmed that there was no distortion of the pedicle omentum through a rigid laparoscope (Fig. 1E and F). Several sutures were placed to fix the omentum and diaphragm. Finally, a total of 6 5-layer cell sheets were implanted on a different area to cover the infarct and border regions, followed by the placement of the pedicle omentum over the sheets. Muscle and skin were closed in layers, and anaesthesia was completed (Video 1). Figure 1: View largeDownload slide (A) A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was placed on the fixed site of the diaphragm, followed by (B) advancement of a flexible gastrointestinal endoscope into the intra-abdominal cavity through the port. (C) A reusable biopsy forceps (FG-47L-1, Olympus, Tokyo, Japan) was introduced via flexible endoscopy. (D) The omentum was grasped, (E) brought back to the pleural cavity via the diaphragm and (F) then the omentum was further mobilized with sufficient volume to cover the left ventricle. Figure 1: View largeDownload slide (A) A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was placed on the fixed site of the diaphragm, followed by (B) advancement of a flexible gastrointestinal endoscope into the intra-abdominal cavity through the port. (C) A reusable biopsy forceps (FG-47L-1, Olympus, Tokyo, Japan) was introduced via flexible endoscopy. (D) The omentum was grasped, (E) brought back to the pleural cavity via the diaphragm and (F) then the omentum was further mobilized with sufficient volume to cover the left ventricle. Video 1 Procedure for transphrenic peritoneoscopy-assisted omentopexy with cell sheet transplantation for damaged heart. Video 1 Procedure for transphrenic peritoneoscopy-assisted omentopexy with cell sheet transplantation for damaged heart. Close Haemodynamic monitoring Haemodynamic (heart rate and invasive arterial pressure via femoral artery) and respiratory (respiratory rate and arterial oxygen saturation by pulse oximetry) parameters were recorded during the surgical procedure. In addition, arterial blood samples were drawn at 5 predefined time points as follows: T0, after anaesthetic induction and stabilization (under two-lung ventilation); T1, after switching from 2- to 1-lung ventilation for the rethoracotomy; T2, after starting to bring the pedicle omentum to the pleural cavity under 1-lung ventilation; T3, after switching from 1- to 2-lung ventilation after TPP-assisted OP and T4, after closing the chest (end of operation). Cardiac magnetic resonance imaging MRI was performed using a 1.5-T scanner (Signa EXCITE XI TwinSpeed; GE Medical Systems, Milwaukee, WI, USA). To assess changes in LV function, imaging was performed 1 day before and 4 weeks after treatment. Short-axis images with an 8-mm slice thickness that included the entire heart were obtained using pulse parameters for cardiac-gated, fast gradient-recalled echo. Axial images were reconstructed using dedicated scanning software. All images were analysed using a workstation (Virtual Place Lexus64; AZE, Tokyo, Japan), which provided LV end-diastolic volume (EDV) and LV end-systolic volume (ESV) values, and then the LV ejection fraction (EF) was calculated with the following formula: LVEF (%) = 100 × (LVEDV−LVESV)/(LVEDV). PET measurements and data analysis To evaluate changes in regional myocardial blood flow (MBF), 13N-ammonia positron emission tomography (PET) measurements were serially obtained under general anaesthesia 1 day before and 3 weeks after treatment using a Headtome V/SET2400W (Shimadzu, Kyoto, Japan). Transmission scanning with an external line source (68Ge–68Ga) was performed for attenuation correction. Twenty-minute PET measurements were initiated at the same time as a bolus administration of 13N-NH3 (300–350 MBq). PET images were reconstructed using a 2D-filtered back projection algorithm with correction for dead time and decay. The image matrix was 128 × 128 × 63, which resulted in a voxel size of 2.0 × 2.0 × 3.125 mm. Regions of interest were semiautomatically placed on the LV myocardium and divided into 17 segments on the summed PET images. A polar map of PET images was generated by setting the maximum value of the intact myocardium as 100%. Changes in MBF are presented as percent change by dividing post-treatment MBF by pretreatment MBF based on the polar map. Angiographic study One month after treatment, a median sternotomy was performed in the supine position to expose the heart under general anaesthesia. For mini pigs that received the experimental treatment (n = 2), a skin incision was extended into the abdomen, followed by a laparotomy, taking care not to injure the pedicle of the omentum. After evaluating the anatomical findings, a catheter (25114110, EV-21G × 150 mm, Hakko Co., Ltd., Nagano, Japan) was inserted via the gastroepiploic artery, followed by systemic heparinization. Each animal was humanely euthanized, and then India ink was selectively injected into the gastroepiploic artery to confirm vessel communication between the pedicle of the omentum and myocardium. The heart was then harvested and fixed in 4% paraformaldehyde. End-points The primary end-point of this study was the feasibility and safety of our TPP-assisted procedure in a myocardial infraction porcine model. The secondary end-points included anatomical evaluations (torsion and/or diaphragmatic hernia) 1 month after treatment, degree of improvement in cardiac function and communication between the pedicle of the omentum and damaged myocardium. RESULTS Haemodynamic parameters during cell therapy plus transphrenic peritoneoscopy-assisted omentopexy Cardiac MRI was performed at 1 month after induction of MI, which showed a dilated LV volume and impaired LVEF in all the mini pigs (n = 4) (Table 1). TPP-assisted OP was accomplished in 2 of the animals with an intra-abdominal pressure ≤8 mmHg within 30 min (22 and 27 min, respectively). The haemodynamic and pulmonary parameters during the procedure are listed in Fig. 2 and Table 2. Table 1: Serial cardiac MRI findings showing changes in LV function Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 LV: left ventricular; LVEDV: left ventricular end-diastolic volume; LVEF: left ventricular ejection fraction; LVESV: left ventricular end-systolic volume; MI: myocardial infarction; MRI: magnetic resonance imaging. Table 1: Serial cardiac MRI findings showing changes in LV function Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 LV: left ventricular; LVEDV: left ventricular end-diastolic volume; LVEF: left ventricular ejection fraction; LVESV: left ventricular end-systolic volume; MI: myocardial infarction; MRI: magnetic resonance imaging. Table 2: Arterial blood gas measurements during procedure T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 BE: base excess; HCO3: bicarbonate; PCO2: partial pressure of carbon dioxide; PO2: partial pressure of oxygen; sO2: oxygen saturation. Table 2: Arterial blood gas measurements during procedure T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 BE: base excess; HCO3: bicarbonate; PCO2: partial pressure of carbon dioxide; PO2: partial pressure of oxygen; sO2: oxygen saturation. Figure 2: View largeDownload slide Haemodynamic and pulmonary parameters during the procedure in the (A) first and (B) second treated mini pigs. (C) Detailed process of TPP-assisted omentopexy. ABG: arterial blood gas; BP: blood pressure; DOB: dobutamine; TPP: transphrenic peritoneoscopy. Figure 2: View largeDownload slide Haemodynamic and pulmonary parameters during the procedure in the (A) first and (B) second treated mini pigs. (C) Detailed process of TPP-assisted omentopexy. ABG: arterial blood gas; BP: blood pressure; DOB: dobutamine; TPP: transphrenic peritoneoscopy. In the first treatment case, there were no substantial changes in systolic and diastolic arterial pressures or arterial oxygen saturation (SpO2). Although heart rate showed an increase along with a modest increase in respiratory rate after switching from 2- to 1-lung ventilation for the rethoracotomy, further deterioration was not observed in these values, and systolic/diastolic blood pressure remained stable. Similarly, the arterial blood gas sample showed a temporary increase in the concentration of carbon dioxide and a decrease in that of oxygen, and these parameters immediately returned to normal levels after switching to 2-lung ventilation following the TPP-assisted OP. In the second case, cardiac MRI findings revealed not only an impaired LV function but also an extremely dilated right ventricle. In addition, we viewed massive peritoneal effusion through the rigid laparoscope, indicating that this animal had severe right and left ventricular failure. The presence of ascites did not allow us to obtain a clear surgical exposure with an intra-abdominal pressure of 4 mmHg, and thus we were forced to increase that up to 8 mmHg. In this case, there was a significant decrease in blood pressure after switching from 2- to 1-lung ventilation for the rethoracotomy, along with a substantial increase in heart rate (e.g. occurrence of atrial fibrillation). Haemodynamics became stable following temporary use of an inotropic agent and switching again from 1- to 2-lung ventilation. The arterial blood gas sample showed a substantial increase in the concentration of carbon dioxide during 1-lung ventilation, whereas that of oxygen remained within normal limits with an arterial oxygen saturation of nearly 100% throughout the procedure. The concentration of carbon dioxide returned to a normal level after switching to 2-lung ventilation following TPP-assisted OP, which resulted in successful extubation and early recovery. Impact of cell therapy plus transphrenic peritoneoscopy-assisted omentopexy on left ventricle function recovery MRI findings of the control group revealed ongoing LV dilatation and deterioration of LV systolic function, whereas those of the treatment group at 1 month after cell sheet transplantation with TPP-assisted OP revealed substantial improvements in cardiac function (Table 1, Fig. 3A–H). Figure 3: View largeDownload slide Representative images of serial cardiac magnetic resonance imaging in (A–D) untreated (control) and (E–H) treated animals. LV: left ventricle; RV: right ventricle. Figure 3: View largeDownload slide Representative images of serial cardiac magnetic resonance imaging in (A–D) untreated (control) and (E–H) treated animals. LV: left ventricle; RV: right ventricle. Impact of cell therapy plus transphrenic peritoneoscopy-assisted omentopexy on myocardial blood flow One month after induction of MI, MBF in the anterior and/or septal territories was substantially decreased when compared with that in the lateral/inferior territories in all 4 animals. Serial PET examinations revealed that relative MBF in the global LV was decreased by 15.3% and 3.4% in the first and second untreated mini pigs, respectively, with comparable changes (decreases) between basal, mid LV and apex (Table 3, Fig. 4A and B). In contrast, that value was substantially increased by 10.2% with comparable changes (increases) across the LV at 1 month after treatment in the first treated mini pig, whereas it remained unchanged in the second treated mini pig (Table 3, Fig. 4C and D). Table 3: Serial PET examinations Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 LV: left ventricle; MBF: myocardial blood flow; PET: position emission tomography. Table 3: Serial PET examinations Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 LV: left ventricle; MBF: myocardial blood flow; PET: position emission tomography. Figure 4: View largeDownload slide Percent change in relative MBF according to LV segment in (A, B) untreated (control) and (C, D) treated animals based on treated animals based on serial PET assessments. LV: left ventricle; MBF: myocardial blood flow. Figure 4: View largeDownload slide Percent change in relative MBF according to LV segment in (A, B) untreated (control) and (C, D) treated animals based on treated animals based on serial PET assessments. LV: left ventricle; MBF: myocardial blood flow. Postmortem examination None of the mini pigs showed mortality related to the procedure or otherwise before planned euthanasia. Necropsy results at 4 weeks after treatment revealed a viable omentum-flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination (Fig. 5A). Selective angiography also revealed that the gastroepiploic artery branches feeding the omentum had expanded into the heart (Fig. 5B, Video 2), whereas histological analysis confirmed vessel communication between the omentum and myocardium (Fig. 5C). In addition, macroscopic findings showed that the omentum tissue was firmly attached to the surface of the left ventricle in the first- and second-treated mini pigs (Fig. 5D). Figure 5: View largeDownload slide (A) Necropsy findings 4 weeks after treatment revealed a viable omentum flap and pedicle (white arrowhead), with no evidence of complications. (B) Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. (C, D) (white arrowheads indicate omentum tissue attached to the surface of the LV). Macroscopic images of heart in second treatment model. (E, F) Histological analysis confirmed vessel communication between the omentum and myocardium (green arrowheads). LV: left ventricle; RV: right ventricle. Figure 5: View largeDownload slide (A) Necropsy findings 4 weeks after treatment revealed a viable omentum flap and pedicle (white arrowhead), with no evidence of complications. (B) Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. (C, D) (white arrowheads indicate omentum tissue attached to the surface of the LV). Macroscopic images of heart in second treatment model. (E, F) Histological analysis confirmed vessel communication between the omentum and myocardium (green arrowheads). LV: left ventricle; RV: right ventricle. Video 2 Necropsy findings at 4 weeks after treatment revealed a viable omentum flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination. Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. Video 2 Necropsy findings at 4 weeks after treatment revealed a viable omentum flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination. Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. Close DISCUSSION Patients requiring an omental flap are usually already ill from a previous complication, thus great care must be taken during omental harvesting to avoid further problems. The present findings demonstrated that cell sheet transplantation combined with TPP-assisted OP was successfully performed at an intra-abdominal pressure <8 mmHg in 2 randomly selected mini pigs with post-MI, one of which showed severe right and left ventricular dysfunction in cardiac MRI results. Notably, when compared with the untreated mini pigs, the mini pigs that received treatment showed evidence of substantial left ventricular function recovery with preserved myocardial perfusion in serial cardiac MRI and NH3-PET findings. A possible advantage of a TPP approach is the relatively low level of intra-abdominal pressure required for intervention within the peritoneal cavity. In laparoscopic or laparoscopic-assisted abdominal surgery, a wide intra-abdominal space is mandatory to maintain a sufficient working space for the use of traditional rigid instruments introduced via separate multiple ports, which usually requires an intra-abdominal pressure level of at least 12 mmHg [15]. On the other hand, a TPP approach does not necessarily require such a wide intra-abdominal space, since a more limited working space is sufficient for instrumentation employed via the flexible endoscope [8–11]. Notably, the concept of TPP-assisted OP is reasonable, as the omentum is located in the most anterior among the abdominal organs, floating on the surface of the intestine. The use of low intraperitoneal pressure might allow for improved cardiopulmonary stability, which is a potential advantage, particularly in critically ill patients. In addition, a flexible endoscope is easier to handle than a conventional rigid laparoscope, even when only limited working space is available in the thoracic cavity, above the diaphragm, below the enlarged heart (Fig. 2A and B). The present favourable results led us to speculate that this approach can be clinically applied not only for patients undergoing cell therapy through a left thoracotomy but also for those who need infection control such as bronchial-pleural fistulas, as well as for patients with a space defect associated with empyema, threatened airway anastomosis, any leakage after an oesophagectomy, a post-sternotomy mediastinal infection or a tracheoesophageal fistula. Another potential advantage of this strategy is that it provides access to treat the heart without the need for a median sternotomy, indicating that our method would be helpful for heart failure patients who have a history of heart surgery (e.g. revascularization). In addition, it would also be useful as sole therapy for treating patients with heart failure secondary to a non-ischaemic aetiology, and surgical revascularization is therefore not indicated. In those cases, the heart could be accessed via a median sternotomy without a substantial increase in risk if a subsequent heart transplantation or implantation of a left ventricular assist device is required in the future following cell therapy with TPP-assisted OP. It is noteworthy that arterial blood gas monitoring showed a temporal but substantial increase in concentration of carbon dioxide and decrease in concentration of oxygen in the treatment animals. However, these findings were unexceptional, as they were seen during 1-lung ventilation for either a re-thoracotomy or TPP-assisted OP, and quickly returned to normal levels after switching to 2-lung ventilation, suggesting that the abnormalities were not directly related to the TPP approach itself and could be controlled by meticulous respiratory management with mechanical ventilation. In addition, changes in arterial blood gas measurements did not significantly affect other haemodynamics, such as blood pressure and heart rate, resulting in successful extubation and early recovery of the animals. Together, our findings suggest that the present TPP approach is feasible, even for cases with severely impaired right heart failure and left heart failure. The omentum is known to play a key role in controlling the spread of inflammation, and it also promotes revascularization, reconstruction and tissue regeneration [1–3, 16]. Results from our previous study that used a rat myocardial infraction model suggest that cell sheet therapy combined with conventional OP (with a laparotomy) improves the hypoxic environment in the transplanted area to a greater degree, potentially enhancing initial cell engraftment and enhancing the paracrine effects induced by the cell sheet [7]. In addition, the present combined treatment is shown to establish functional structurally mature vessels in the ischaemic myocardium, subsequently leading to therapeutic improvement related to blood perfusion. Along with these favourable effects, combined treatment had greater impact not only on LV, haemodynamic and endothelial functions but also on functional capacity when compared with a single treatment (i.e. cell sheet therapy alone). Similarly, Shudo et al. [6] used a mini pig MI model and observed that improvement in cardiac function induced by cell sheet therapy combined with conventional OP (with laparotomy) was greater than that with cell sheet therapy alone, which was maintained for at least to 2 months after treatment. Although the current experimental study was not designed to have statistical power to demonstrate the therapeutic effects of this procedure, our findings of substantial improvement in cardiac function and preservation of MBF in the treatment group are consistent with those presented in our previous studies [6, 7]. Furthermore, they indicate that TPP-assisted OP may achieve a therapeutic effect equivalent to that of conventional OP when combined with cell therapy. Nevertheless, future studies with a more sophisticated design are warranted to confirm our results. Limitations The main limitation of this study is the small number of animals, thus any conclusions are limited. Although this experimental study determined the feasibility of TPP-assisted OP in a large animal model with severe heart failure, additional investigations with a larger sample size are definitely required. CONCLUSION This TPP approach was shown to be feasible and safe with a low-pressure pneumoperitoneum, while the omentum flap was found to be durable and had a synergetic impact on cell sheet implantation. This successful combination of techniques may provide for less-invasive endoscopic intervention and regenerative therapy, particularly in critically ill patients. Funding This work was supported by a J-CASE (Japan Consortium for Advanced Surgical Endoscopy) Research Grant in 2012. Conflict of interest: none declared. REFERENCES 1 O’Shaugnessy L. Surgical treatment of cardiac ischemia . Lancet 1937 ; 232 : 185 – 94 . Google Scholar CrossRef Search ADS 2 Shrager JB , Wain JC , Wright CD , Donahue DM , Vlahakes GJ , Moncure AC et al. . Omentum is highly effective in the management of complex cardiothoracic surgical problems . J Thorac Cardiovasc Surg 2003 ; 125 : 526 – 32 . Google Scholar CrossRef Search ADS PubMed 3 Takaba K , Jiang C , Nemoto S , Saji Y , Ikeda T , Urayama S et al. . A combination of omental flap and growth factor therapy induces arteriogenesis and increases myocardial perfusion in chronic myocardial ischemia: evolving concept of biologic coronary artery bypass grafting . J Thorac Cardiovasc Surg 2006 ; 132 : 891 – 9 . Google Scholar CrossRef Search ADS PubMed 4 D'Andrilli A , Ibrahim M , Andreetti C , Ciccone AM , Venuta F , Rendina EA. Transdiaphragmatic harvesting of the omentum through thoracotomy for bronchial stump reinforcement . Ann Thorac Surg 2009 ; 88 : 212 – 15 . Google Scholar CrossRef Search ADS PubMed 5 Kawamura M , Miyagawa S , Fukushima S , Saito A , Miki K , Ito E et al. . Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart . Circulation 2013 ; 128 : S87 – 94 . Google Scholar CrossRef Search ADS PubMed 6 Shudo Y , Miyagawa S , Fukushima S , Saito A , Shimizu T , Okano T et al. . Novel regenerative therapy using cell-sheet covered with omentum flap delivers a huge number of cells in a porcine myocardial infarction model . J Thorac Cardiovasc Surg 2011 ; 142 : 1188 – 96 . Google Scholar CrossRef Search ADS PubMed 7 Kainuma S , Miyagawa S , Fukushima S , Pearson J , Chen YC , Saito A et al. . Cell-sheet therapy with omentopexy promotes arteriogenesis and improves coronary circulation physiology in failing heart . Mol Ther 2015 ; 23 : 374 – 86 . Google Scholar CrossRef Search ADS PubMed 8 von Delius S , Schorn A , Grimm M , Schneider A , Wilhelm D , Schuster T et al. . Natural-orifice transluminal endoscopic surgery: low-pressure pneumoperitoneum is sufficient and is associated with an improved cardiopulmonary response (PressurePig Study) . Endoscopy 2011 ; 43 : 808 – 15 . Google Scholar CrossRef Search ADS PubMed 9 Higashi S , Nakajima K , Miyazaki Y , Makino T , Takahashi T , Kurokawa Y et al. . A case of transvaginal NOTES partial gastrectomy using new techniques and devices . Surg Case Rep 2015 ; 1 : 96. Google Scholar CrossRef Search ADS PubMed 10 Nakajima K , Takahashi T , Souma Y , Shinzaki S , Yamada T , Yoshio T et al. . Transvaginal endoscopic partial gastrectomy in porcine models: the role of an extra endoscope for gastric control . Surg Endosc 2008 ; 22 : 2733 – 6 . Google Scholar CrossRef Search ADS PubMed 11 Nakajima K , Nishida T , Takahashi T , Souma Y , Hara J , Yamada T et al. . Partial gastrectomy using natural orifice transluminal endoscopic surgery (NOTES) for gastric submucosal tumors: early experience in humans . Surg Endosc 2009 ; 23 : 2650 – 5 . Google Scholar CrossRef Search ADS PubMed 12 Kraitchman DL , Heldman AW , Atalar E , Amado LC , Martin BJ , Pittenger MF et al. . In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction . Circulation 2003 ; 107 : 2290 – 3 . Google Scholar CrossRef Search ADS PubMed 13 Miyagawa S , Sawa Y , Sakakida S , Taketani S , Kondoh H , Memon IA et al. . Tissue cardiomyoplasty using bioengineered contractile cardiomyocyte sheets to repair damaged myocardium: their integration with recipient myocardium . Transplantation 2005 ; 80 : 1586 – 95 . Google Scholar CrossRef Search ADS PubMed 14 Shimizu T , Sekine H , Yang J , Isoi Y , Yamato M , Kikuchi A et al. . Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues . FASEB J 2006 ; 20 : 708 – 10 . Google Scholar CrossRef Search ADS PubMed 15 Neudecker J , Sauerland S , Neugebauer E , Bergamaschi R , Bonjer HJ , Cuschieri A et al. . The European Association for Endoscopic Surgery clinical practice guideline on the pneumoperitoneum for laparoscopic surgery . Surg Endosc 2002 ; 16 : 1121 – 43 . Google Scholar CrossRef Search ADS PubMed 16 MacArthur JW , Goldstone AB , Cohen JE , Hiesinger W , Woo YJ. Cell transplantation in heart failure: where do we stand in 2016? Eur J Cardiothorac Surg 2016 ; 50 : 396 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Interactive CardioVascular and Thoracic Surgery Oxford University Press

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
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Abstract

Abstract OBJECTIVES We previously reported that cell sheet transplantation combined with an omentopexy (OP) procedure is more effective for repairing heart damage when compared with cell sheet transplantation alone. However, a simultaneous (conventional) laparotomy as part of the OP may adversely affect the general condition of critically ill heart failure patients who would otherwise benefit from cell sheet transplantation, which is a paradox to be reconciled before this treatment can be applied in a clinical setting. We devised a novel endoscopic approach termed ‘transphrenic peritoneoscopy’ (TPP) for minimal access to abdominal organs from the thoracic cavity. Herein, we evaluated the feasibility and safety of TPP with an OP in a porcine myocardial infarction model. METHODS Myocardial infarction was induced in 4 mini pigs by placing an ameroid constrictor around the left anterior descending artery. One month later, a left thoracotomy was performed in 2 randomly selected mini pigs, and a laparoscopic port was placed on the left diaphragm to gain access into the abdominal cavity. Using a low-pressure pneumoperitoneum, a flexible gastrointestinal endoscope was advanced, then the omentum was partially grasped with endoscopic forceps and brought back into the thoracic cavity via the diaphragm. Skeletal myoblast cell sheets were then implanted over the impaired myocardium, followed by placing the omentum over the sheets. RESULTS TPP-assisted OP was accomplished in 2 post-myocardial infarction mini pigs with severe heart failure with an intra-abdominal pressure ≤8 mmHg within 30 min (22 and 27 min, respectively). Necropsy findings revealed a viable omentum flap and pedicle in both animals, with no evidence of procedure-related complications. Angiographic and histological analyses confirmed vessel communication between the omentum and the left ventricle. CONCLUSIONS Our TPP approach was shown to be feasible and safe with a low-pressure pneumoperitoneum, while the omentum flap was durable. This successful combination of techniques may provide less-invasive endoscopic intervention and regenerative therapy. Cell therapy, Laparotomy, Omentopexy, Omentum INTRODUCTION The omentum has historically been used for surgical revascularization in patients with ischaemic heart disease and is also known to release a number of angiogenic cytokines and attenuate inflammation [1, 2]. In addition, the gastroepiploic artery involved in the omentum flap plays an important role as an extracardiac blood source with high perfusion capacity for developing effective collateral vessels in the treatment for advanced coronary artery disease [3]. Because of its unique characteristics, an omentopexy (OP) procedure (transposition into the thoracic cavity through the diaphragm) has been clinically applied in the field of thoracic surgery to control infections, such as postoperative mediastinitis and empyema, as well as for cases of abdominal surgery to repair and prevent infections due to gastrointestinal anastomotic leakage and/or perforation [1, 4]. On the basis of this background, we have recently applied the omentum flap to the field of regeneration therapy and successfully established a unique therapeutic method that utilizes cell sheet transplantation combined with an OP in both small and large heart failure animal models [5–7]. Recently, we reported that this cell sheet transplantation method in combination with an OP was more effective for repairing a damaged heart when compared with cell sheet transplantation alone. However, a simultaneous (conventional) laparotomy as part of an OP may adversely affect the general condition of critically ill heart failure patients who would otherwise benefit from cell sheet transplantation through a left thoracotomy, which is a contradiction to be reconciled before this combined treatment can be applied in clinical settings. Although a laparoscopy-assisted approach for abdominal organs may be an alternative to a conventional laparotomy, the possibility that a pneumoperitoneum under usual intra-abdominal pressure (12–15 mmHg) causes a considerable decrease in venous return cannot be ignored, as it subsequently may lead to low cardiac output syndrome in patients with refractory heart failure [8]. To overcome the procedure-related issues noted earlier, we have devised a novel approach termed ‘transphrenic peritoneoscopy’ (TPP) for minimal access to abdominal organs from the thoracic cavity via a port placed on the diaphragm with a flexible gastrointestinal endoscope. This procedure is derived from the concept of ‘natural orifice transluminal endoscopic surgery’, which is a surgical technique whereby ‘scarless’ abdominal operations can be performed by inserting the endoscope through a natural orifice (e.g. mouth, urethra and anus) and entering the peritoneal cavity by making an incision on the luminal wall (e.g. stomach, vagina, bladder and colon) [9–11]. Herein, we evaluated the feasibility and safety of TPP-assisted OP in a porcine myocardial infarction (MI) model. In addition, we assessed its potential effectiveness for enhancing cell-sheet-mediated regenerative medicine. To the best of our knowledge, this is the first preclinical report on TPP, or transphrenic natural orifice transluminal endoscopic surgery, presented in the surgical literature. MATERIALS AND METHODS All experimental procedures were approved by our institutional ethics committee. Animal care was performed humanely in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Animal Resources and published by the National Institutes of Health (publication number 85-23, revised 1996). Study protocol MI was induced in 4 female mini pigs (8–10-month old, Kagoshima Miniature Swine Research Center, Kagoshima, Japan) each weighing 20–25 kg. Four weeks after MI, the animals were randomly divided into either the treatment (cell sheet transplantation plus TPP-assisted OP) or the control (non-treatment) group (n = 2 each). To assess the effect of treatment on LV volumes and function, electrocardiogram (ECG)-gated cardiac magnetic resonance imaging (MRI) was performed approximately 1 month after MI (e.g. 1 day prior to treatment) and 1 month after treatment with an 8-channel cardiac coil wrapped around the chest wall [12]. At the end-point of this study (1 month after treatment), the animals were humanely euthanized for histological and angiographical analyses of heart and omentum tissues. Creation of autologous skeletal muscle cell sheets Autologous skeletal muscle tissue weighing approximately 10–15 g was removed from the quadriceps femoris muscle, and then purified autologous skeletal muscle cells were cultured for 3 weeks to prepare for implantation, as previously described [6, 13, 14]. Briefly, the cells were incubated in 60-mm temperature-responsive culture dishes (UpCell®; Cellseed, Tokyo, Japan) at 37°C in 5% CO2 atmosphere for 24 h with the cell numbers adjusted to 1.5 × 107 cells per dish. The dishes were then transferred to another incubator set at 20°C for 1 h to release the cultured cells as an intact cell sheet. Using this protocol, skeletal muscle cells were spontaneously detached from the plate as a free-floating monolayer cell sheet. Generation of porcine chronic heart failure model Each mini pig was anaesthetized with an intravenous administration of ketamine hydrochloride (10 mg/kg; DAIICHI SANKYO, Tokyo, Japan) and xylazine (2 mg/kg; Bayer HealthCare, Leverkusen, Germany) for endotracheal intubation, and then anaesthesia was maintained with inhaled sevoflurane (2%). The pericardial space was exposed by a left thoracotomy through the 4th intercostal space. The distal portion of the left anterior descending coronary artery was directly ligated as ischaemic preconditioning to reduce the occurrence of lethal ventricular arrhythmia, followed by placement of an ameroid constrictor (COR-2.50-SS; Research Instruments SW, Escondido, CA, USA) around the left anterior descending coronary artery just distal to the left circumflex coronary artery branching [6, 13]. The muscle and skin were closed in layers, then anaesthesia was completed, and the animal was allowed to recover alone in a temperature-controlled cage. This technique produced an ischaemic cardiomyopathy model that reflected clinical relevance and was considered appropriate for preclinical studies with minimal procedure-related mortality. Cell sheet transplantation combined with transphrenic peritoneoscopy-assisted omentopexy Under general anaesthesia and double-lumen endotracheal intubation, a 12-mm standard laparoscopic port was created at the umbilical site with the animal in the supine position, and a standard laparoscopic CO2 insufflator (UHI-3, Olympus Corporation, Tokyo, Japan) was used to initiate and maintain the pneumoperitoneum with a pressure of 4 mmHg to check for any obvious abnormality in the abdominal cavity. After moving to a right lateral decubitus position, a second left thoracotomy was made under 1-lung ventilation, and the pericardium was incised to ensure a wider visual field of the left ventricle. The optimal delivery route of the pedicle omentum was determined by viewing directly from the left thoracotomy and intra-abdominal cavity through a rigid laparoscope. A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was then placed on the fixed site of the diaphragm to access the abdominal cavity, while paying close attention to not injure abdominal organs (Fig. 1A). A flexible gastrointestinal endoscope (GIF-Q260J, Olympus Medical Systems, Tokyo, Japan) was advanced into the intra-abdominal cavity through the port under the pneumoperitoneum with a pressure of 4 mmHg (up to 8 mmHg maximum if required) (Fig. 1B and C). The omentum was then identified and grasped with a reusable biopsy forceps (FG-47l-1, Olympus, Tokyo, Japan) and brought back to the pleural cavity via the diaphragm where the port was placed under 1-lung ventilation (Fig. 1D). The omentum grasped by the forceps was further mobilized with sufficient volume to cover the left ventricle, during which we confirmed that there was no distortion of the pedicle omentum through a rigid laparoscope (Fig. 1E and F). Several sutures were placed to fix the omentum and diaphragm. Finally, a total of 6 5-layer cell sheets were implanted on a different area to cover the infarct and border regions, followed by the placement of the pedicle omentum over the sheets. Muscle and skin were closed in layers, and anaesthesia was completed (Video 1). Figure 1: View largeDownload slide (A) A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was placed on the fixed site of the diaphragm, followed by (B) advancement of a flexible gastrointestinal endoscope into the intra-abdominal cavity through the port. (C) A reusable biopsy forceps (FG-47L-1, Olympus, Tokyo, Japan) was introduced via flexible endoscopy. (D) The omentum was grasped, (E) brought back to the pleural cavity via the diaphragm and (F) then the omentum was further mobilized with sufficient volume to cover the left ventricle. Figure 1: View largeDownload slide (A) A laparoscopic port with a radially expandable sleeve (VersaStep Plus 12 mm) was placed on the fixed site of the diaphragm, followed by (B) advancement of a flexible gastrointestinal endoscope into the intra-abdominal cavity through the port. (C) A reusable biopsy forceps (FG-47L-1, Olympus, Tokyo, Japan) was introduced via flexible endoscopy. (D) The omentum was grasped, (E) brought back to the pleural cavity via the diaphragm and (F) then the omentum was further mobilized with sufficient volume to cover the left ventricle. Video 1 Procedure for transphrenic peritoneoscopy-assisted omentopexy with cell sheet transplantation for damaged heart. Video 1 Procedure for transphrenic peritoneoscopy-assisted omentopexy with cell sheet transplantation for damaged heart. Close Haemodynamic monitoring Haemodynamic (heart rate and invasive arterial pressure via femoral artery) and respiratory (respiratory rate and arterial oxygen saturation by pulse oximetry) parameters were recorded during the surgical procedure. In addition, arterial blood samples were drawn at 5 predefined time points as follows: T0, after anaesthetic induction and stabilization (under two-lung ventilation); T1, after switching from 2- to 1-lung ventilation for the rethoracotomy; T2, after starting to bring the pedicle omentum to the pleural cavity under 1-lung ventilation; T3, after switching from 1- to 2-lung ventilation after TPP-assisted OP and T4, after closing the chest (end of operation). Cardiac magnetic resonance imaging MRI was performed using a 1.5-T scanner (Signa EXCITE XI TwinSpeed; GE Medical Systems, Milwaukee, WI, USA). To assess changes in LV function, imaging was performed 1 day before and 4 weeks after treatment. Short-axis images with an 8-mm slice thickness that included the entire heart were obtained using pulse parameters for cardiac-gated, fast gradient-recalled echo. Axial images were reconstructed using dedicated scanning software. All images were analysed using a workstation (Virtual Place Lexus64; AZE, Tokyo, Japan), which provided LV end-diastolic volume (EDV) and LV end-systolic volume (ESV) values, and then the LV ejection fraction (EF) was calculated with the following formula: LVEF (%) = 100 × (LVEDV−LVESV)/(LVEDV). PET measurements and data analysis To evaluate changes in regional myocardial blood flow (MBF), 13N-ammonia positron emission tomography (PET) measurements were serially obtained under general anaesthesia 1 day before and 3 weeks after treatment using a Headtome V/SET2400W (Shimadzu, Kyoto, Japan). Transmission scanning with an external line source (68Ge–68Ga) was performed for attenuation correction. Twenty-minute PET measurements were initiated at the same time as a bolus administration of 13N-NH3 (300–350 MBq). PET images were reconstructed using a 2D-filtered back projection algorithm with correction for dead time and decay. The image matrix was 128 × 128 × 63, which resulted in a voxel size of 2.0 × 2.0 × 3.125 mm. Regions of interest were semiautomatically placed on the LV myocardium and divided into 17 segments on the summed PET images. A polar map of PET images was generated by setting the maximum value of the intact myocardium as 100%. Changes in MBF are presented as percent change by dividing post-treatment MBF by pretreatment MBF based on the polar map. Angiographic study One month after treatment, a median sternotomy was performed in the supine position to expose the heart under general anaesthesia. For mini pigs that received the experimental treatment (n = 2), a skin incision was extended into the abdomen, followed by a laparotomy, taking care not to injure the pedicle of the omentum. After evaluating the anatomical findings, a catheter (25114110, EV-21G × 150 mm, Hakko Co., Ltd., Nagano, Japan) was inserted via the gastroepiploic artery, followed by systemic heparinization. Each animal was humanely euthanized, and then India ink was selectively injected into the gastroepiploic artery to confirm vessel communication between the pedicle of the omentum and myocardium. The heart was then harvested and fixed in 4% paraformaldehyde. End-points The primary end-point of this study was the feasibility and safety of our TPP-assisted procedure in a myocardial infraction porcine model. The secondary end-points included anatomical evaluations (torsion and/or diaphragmatic hernia) 1 month after treatment, degree of improvement in cardiac function and communication between the pedicle of the omentum and damaged myocardium. RESULTS Haemodynamic parameters during cell therapy plus transphrenic peritoneoscopy-assisted omentopexy Cardiac MRI was performed at 1 month after induction of MI, which showed a dilated LV volume and impaired LVEF in all the mini pigs (n = 4) (Table 1). TPP-assisted OP was accomplished in 2 of the animals with an intra-abdominal pressure ≤8 mmHg within 30 min (22 and 27 min, respectively). The haemodynamic and pulmonary parameters during the procedure are listed in Fig. 2 and Table 2. Table 1: Serial cardiac MRI findings showing changes in LV function Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 LV: left ventricular; LVEDV: left ventricular end-diastolic volume; LVEF: left ventricular ejection fraction; LVESV: left ventricular end-systolic volume; MI: myocardial infarction; MRI: magnetic resonance imaging. Table 1: Serial cardiac MRI findings showing changes in LV function Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 Control 1 Control 2 Treatment 1 Treatment 2 1 month after MI induction  LVEDV (ml) 41.0 39.5 44.8 48.9  LVESV (ml) 24.9 24.0 27.7 31.6  LVEF (%) 39.4 39.2 38.2 35.4 1 month after treatment (2 months after MI induction)  LVEDV (ml) 57.2 49.1 40.7 43.8  LVESV (ml) 39.5 33.5 20.6 24.4  LVEF (%) 31.0 31.6 49.2 44.3 LV: left ventricular; LVEDV: left ventricular end-diastolic volume; LVEF: left ventricular ejection fraction; LVESV: left ventricular end-systolic volume; MI: myocardial infarction; MRI: magnetic resonance imaging. Table 2: Arterial blood gas measurements during procedure T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 BE: base excess; HCO3: bicarbonate; PCO2: partial pressure of carbon dioxide; PO2: partial pressure of oxygen; sO2: oxygen saturation. Table 2: Arterial blood gas measurements during procedure T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 T0 T1 T2 T3 T4 Ventilator setting 2 Lungs 1 Lung 1 Lung 2 Lungs 2 Lungs Case 1  pH 7.546 7.366 7.247 7.485 7.533  pCO2 (mmHg) 33.5 44.8 54.9 36.3 35.3  pO2 (mmHg) 506 106 84 472 529  BE (mmol/l) 7 0 2 4 7  HCO3 (mmol/l) 29 25.7 28.4 27.3 29.7  sO2 (%) 100 98 95 100 100 Case 2  pH 7.584 7.335 7.273 7.379 7.468  pCO2 (mmHg) 29.8 59.2 72.7 54.8 41.3  pO2 (mmHg) 364 229 233 149 209  BE (mmol/l) 6 6 7 7 6  HCO3 (mmol/l) 28.2 31.6 33.6 32.3 29.9  sO2 (%) 100 100 100 99 100 BE: base excess; HCO3: bicarbonate; PCO2: partial pressure of carbon dioxide; PO2: partial pressure of oxygen; sO2: oxygen saturation. Figure 2: View largeDownload slide Haemodynamic and pulmonary parameters during the procedure in the (A) first and (B) second treated mini pigs. (C) Detailed process of TPP-assisted omentopexy. ABG: arterial blood gas; BP: blood pressure; DOB: dobutamine; TPP: transphrenic peritoneoscopy. Figure 2: View largeDownload slide Haemodynamic and pulmonary parameters during the procedure in the (A) first and (B) second treated mini pigs. (C) Detailed process of TPP-assisted omentopexy. ABG: arterial blood gas; BP: blood pressure; DOB: dobutamine; TPP: transphrenic peritoneoscopy. In the first treatment case, there were no substantial changes in systolic and diastolic arterial pressures or arterial oxygen saturation (SpO2). Although heart rate showed an increase along with a modest increase in respiratory rate after switching from 2- to 1-lung ventilation for the rethoracotomy, further deterioration was not observed in these values, and systolic/diastolic blood pressure remained stable. Similarly, the arterial blood gas sample showed a temporary increase in the concentration of carbon dioxide and a decrease in that of oxygen, and these parameters immediately returned to normal levels after switching to 2-lung ventilation following the TPP-assisted OP. In the second case, cardiac MRI findings revealed not only an impaired LV function but also an extremely dilated right ventricle. In addition, we viewed massive peritoneal effusion through the rigid laparoscope, indicating that this animal had severe right and left ventricular failure. The presence of ascites did not allow us to obtain a clear surgical exposure with an intra-abdominal pressure of 4 mmHg, and thus we were forced to increase that up to 8 mmHg. In this case, there was a significant decrease in blood pressure after switching from 2- to 1-lung ventilation for the rethoracotomy, along with a substantial increase in heart rate (e.g. occurrence of atrial fibrillation). Haemodynamics became stable following temporary use of an inotropic agent and switching again from 1- to 2-lung ventilation. The arterial blood gas sample showed a substantial increase in the concentration of carbon dioxide during 1-lung ventilation, whereas that of oxygen remained within normal limits with an arterial oxygen saturation of nearly 100% throughout the procedure. The concentration of carbon dioxide returned to a normal level after switching to 2-lung ventilation following TPP-assisted OP, which resulted in successful extubation and early recovery. Impact of cell therapy plus transphrenic peritoneoscopy-assisted omentopexy on left ventricle function recovery MRI findings of the control group revealed ongoing LV dilatation and deterioration of LV systolic function, whereas those of the treatment group at 1 month after cell sheet transplantation with TPP-assisted OP revealed substantial improvements in cardiac function (Table 1, Fig. 3A–H). Figure 3: View largeDownload slide Representative images of serial cardiac magnetic resonance imaging in (A–D) untreated (control) and (E–H) treated animals. LV: left ventricle; RV: right ventricle. Figure 3: View largeDownload slide Representative images of serial cardiac magnetic resonance imaging in (A–D) untreated (control) and (E–H) treated animals. LV: left ventricle; RV: right ventricle. Impact of cell therapy plus transphrenic peritoneoscopy-assisted omentopexy on myocardial blood flow One month after induction of MI, MBF in the anterior and/or septal territories was substantially decreased when compared with that in the lateral/inferior territories in all 4 animals. Serial PET examinations revealed that relative MBF in the global LV was decreased by 15.3% and 3.4% in the first and second untreated mini pigs, respectively, with comparable changes (decreases) between basal, mid LV and apex (Table 3, Fig. 4A and B). In contrast, that value was substantially increased by 10.2% with comparable changes (increases) across the LV at 1 month after treatment in the first treated mini pig, whereas it remained unchanged in the second treated mini pig (Table 3, Fig. 4C and D). Table 3: Serial PET examinations Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 LV: left ventricle; MBF: myocardial blood flow; PET: position emission tomography. Table 3: Serial PET examinations Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 Area Control 1 Control 2 Treatment 1 Treatment 2 Pretreatment relative MBF (%)  Basal LV (Segments 1–6) 80.6 75.8 78.2 70.0  Mid LV (Segments 7–12) 77.0 84.4 79.3 78.9  Apex (Segments 13–17) 57.3 77.2 58.8 59.2 Post-treatment relative MBF (%)  Basal LV (Segments 1–6) 71.8 72.9 84.3 71.6  Mid LV (Segments 7–12) 65.4 82.2 85.8 76.6  Apex (Segments 13–17) 46.8 73.9 67.1 59.0 Percent change (post/pre)  Basal LV (Segments 1–6) −11.3 −3.7 8.6 2.4  Mid LV (Segments 7–12) −16.0 −2.6 8.7 −2.9  Apex (Segments 13–17) −19.2 −4.2 13.9 −0.6  Global LV −15.3 −3.4 10.2 −0.3 LV: left ventricle; MBF: myocardial blood flow; PET: position emission tomography. Figure 4: View largeDownload slide Percent change in relative MBF according to LV segment in (A, B) untreated (control) and (C, D) treated animals based on treated animals based on serial PET assessments. LV: left ventricle; MBF: myocardial blood flow. Figure 4: View largeDownload slide Percent change in relative MBF according to LV segment in (A, B) untreated (control) and (C, D) treated animals based on treated animals based on serial PET assessments. LV: left ventricle; MBF: myocardial blood flow. Postmortem examination None of the mini pigs showed mortality related to the procedure or otherwise before planned euthanasia. Necropsy results at 4 weeks after treatment revealed a viable omentum-flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination (Fig. 5A). Selective angiography also revealed that the gastroepiploic artery branches feeding the omentum had expanded into the heart (Fig. 5B, Video 2), whereas histological analysis confirmed vessel communication between the omentum and myocardium (Fig. 5C). In addition, macroscopic findings showed that the omentum tissue was firmly attached to the surface of the left ventricle in the first- and second-treated mini pigs (Fig. 5D). Figure 5: View largeDownload slide (A) Necropsy findings 4 weeks after treatment revealed a viable omentum flap and pedicle (white arrowhead), with no evidence of complications. (B) Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. (C, D) (white arrowheads indicate omentum tissue attached to the surface of the LV). Macroscopic images of heart in second treatment model. (E, F) Histological analysis confirmed vessel communication between the omentum and myocardium (green arrowheads). LV: left ventricle; RV: right ventricle. Figure 5: View largeDownload slide (A) Necropsy findings 4 weeks after treatment revealed a viable omentum flap and pedicle (white arrowhead), with no evidence of complications. (B) Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. (C, D) (white arrowheads indicate omentum tissue attached to the surface of the LV). Macroscopic images of heart in second treatment model. (E, F) Histological analysis confirmed vessel communication between the omentum and myocardium (green arrowheads). LV: left ventricle; RV: right ventricle. Video 2 Necropsy findings at 4 weeks after treatment revealed a viable omentum flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination. Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. Video 2 Necropsy findings at 4 weeks after treatment revealed a viable omentum flap and pedicle, with no evidence of ileus, bowel obstruction, gastric outlet obstruction, diaphragmatic hernia or peritoneal contamination. Selective angiography findings revealed that gastroepiploic artery branches feeding the omentum had expanded into the heart. Close DISCUSSION Patients requiring an omental flap are usually already ill from a previous complication, thus great care must be taken during omental harvesting to avoid further problems. The present findings demonstrated that cell sheet transplantation combined with TPP-assisted OP was successfully performed at an intra-abdominal pressure <8 mmHg in 2 randomly selected mini pigs with post-MI, one of which showed severe right and left ventricular dysfunction in cardiac MRI results. Notably, when compared with the untreated mini pigs, the mini pigs that received treatment showed evidence of substantial left ventricular function recovery with preserved myocardial perfusion in serial cardiac MRI and NH3-PET findings. A possible advantage of a TPP approach is the relatively low level of intra-abdominal pressure required for intervention within the peritoneal cavity. In laparoscopic or laparoscopic-assisted abdominal surgery, a wide intra-abdominal space is mandatory to maintain a sufficient working space for the use of traditional rigid instruments introduced via separate multiple ports, which usually requires an intra-abdominal pressure level of at least 12 mmHg [15]. On the other hand, a TPP approach does not necessarily require such a wide intra-abdominal space, since a more limited working space is sufficient for instrumentation employed via the flexible endoscope [8–11]. Notably, the concept of TPP-assisted OP is reasonable, as the omentum is located in the most anterior among the abdominal organs, floating on the surface of the intestine. The use of low intraperitoneal pressure might allow for improved cardiopulmonary stability, which is a potential advantage, particularly in critically ill patients. In addition, a flexible endoscope is easier to handle than a conventional rigid laparoscope, even when only limited working space is available in the thoracic cavity, above the diaphragm, below the enlarged heart (Fig. 2A and B). The present favourable results led us to speculate that this approach can be clinically applied not only for patients undergoing cell therapy through a left thoracotomy but also for those who need infection control such as bronchial-pleural fistulas, as well as for patients with a space defect associated with empyema, threatened airway anastomosis, any leakage after an oesophagectomy, a post-sternotomy mediastinal infection or a tracheoesophageal fistula. Another potential advantage of this strategy is that it provides access to treat the heart without the need for a median sternotomy, indicating that our method would be helpful for heart failure patients who have a history of heart surgery (e.g. revascularization). In addition, it would also be useful as sole therapy for treating patients with heart failure secondary to a non-ischaemic aetiology, and surgical revascularization is therefore not indicated. In those cases, the heart could be accessed via a median sternotomy without a substantial increase in risk if a subsequent heart transplantation or implantation of a left ventricular assist device is required in the future following cell therapy with TPP-assisted OP. It is noteworthy that arterial blood gas monitoring showed a temporal but substantial increase in concentration of carbon dioxide and decrease in concentration of oxygen in the treatment animals. However, these findings were unexceptional, as they were seen during 1-lung ventilation for either a re-thoracotomy or TPP-assisted OP, and quickly returned to normal levels after switching to 2-lung ventilation, suggesting that the abnormalities were not directly related to the TPP approach itself and could be controlled by meticulous respiratory management with mechanical ventilation. In addition, changes in arterial blood gas measurements did not significantly affect other haemodynamics, such as blood pressure and heart rate, resulting in successful extubation and early recovery of the animals. Together, our findings suggest that the present TPP approach is feasible, even for cases with severely impaired right heart failure and left heart failure. The omentum is known to play a key role in controlling the spread of inflammation, and it also promotes revascularization, reconstruction and tissue regeneration [1–3, 16]. Results from our previous study that used a rat myocardial infraction model suggest that cell sheet therapy combined with conventional OP (with a laparotomy) improves the hypoxic environment in the transplanted area to a greater degree, potentially enhancing initial cell engraftment and enhancing the paracrine effects induced by the cell sheet [7]. In addition, the present combined treatment is shown to establish functional structurally mature vessels in the ischaemic myocardium, subsequently leading to therapeutic improvement related to blood perfusion. Along with these favourable effects, combined treatment had greater impact not only on LV, haemodynamic and endothelial functions but also on functional capacity when compared with a single treatment (i.e. cell sheet therapy alone). Similarly, Shudo et al. [6] used a mini pig MI model and observed that improvement in cardiac function induced by cell sheet therapy combined with conventional OP (with laparotomy) was greater than that with cell sheet therapy alone, which was maintained for at least to 2 months after treatment. Although the current experimental study was not designed to have statistical power to demonstrate the therapeutic effects of this procedure, our findings of substantial improvement in cardiac function and preservation of MBF in the treatment group are consistent with those presented in our previous studies [6, 7]. Furthermore, they indicate that TPP-assisted OP may achieve a therapeutic effect equivalent to that of conventional OP when combined with cell therapy. Nevertheless, future studies with a more sophisticated design are warranted to confirm our results. Limitations The main limitation of this study is the small number of animals, thus any conclusions are limited. Although this experimental study determined the feasibility of TPP-assisted OP in a large animal model with severe heart failure, additional investigations with a larger sample size are definitely required. CONCLUSION This TPP approach was shown to be feasible and safe with a low-pressure pneumoperitoneum, while the omentum flap was found to be durable and had a synergetic impact on cell sheet implantation. This successful combination of techniques may provide for less-invasive endoscopic intervention and regenerative therapy, particularly in critically ill patients. Funding This work was supported by a J-CASE (Japan Consortium for Advanced Surgical Endoscopy) Research Grant in 2012. Conflict of interest: none declared. REFERENCES 1 O’Shaugnessy L. Surgical treatment of cardiac ischemia . Lancet 1937 ; 232 : 185 – 94 . Google Scholar CrossRef Search ADS 2 Shrager JB , Wain JC , Wright CD , Donahue DM , Vlahakes GJ , Moncure AC et al. . Omentum is highly effective in the management of complex cardiothoracic surgical problems . J Thorac Cardiovasc Surg 2003 ; 125 : 526 – 32 . Google Scholar CrossRef Search ADS PubMed 3 Takaba K , Jiang C , Nemoto S , Saji Y , Ikeda T , Urayama S et al. . A combination of omental flap and growth factor therapy induces arteriogenesis and increases myocardial perfusion in chronic myocardial ischemia: evolving concept of biologic coronary artery bypass grafting . J Thorac Cardiovasc Surg 2006 ; 132 : 891 – 9 . Google Scholar CrossRef Search ADS PubMed 4 D'Andrilli A , Ibrahim M , Andreetti C , Ciccone AM , Venuta F , Rendina EA. Transdiaphragmatic harvesting of the omentum through thoracotomy for bronchial stump reinforcement . Ann Thorac Surg 2009 ; 88 : 212 – 15 . Google Scholar CrossRef Search ADS PubMed 5 Kawamura M , Miyagawa S , Fukushima S , Saito A , Miki K , Ito E et al. . Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart . Circulation 2013 ; 128 : S87 – 94 . Google Scholar CrossRef Search ADS PubMed 6 Shudo Y , Miyagawa S , Fukushima S , Saito A , Shimizu T , Okano T et al. . Novel regenerative therapy using cell-sheet covered with omentum flap delivers a huge number of cells in a porcine myocardial infarction model . J Thorac Cardiovasc Surg 2011 ; 142 : 1188 – 96 . Google Scholar CrossRef Search ADS PubMed 7 Kainuma S , Miyagawa S , Fukushima S , Pearson J , Chen YC , Saito A et al. . Cell-sheet therapy with omentopexy promotes arteriogenesis and improves coronary circulation physiology in failing heart . Mol Ther 2015 ; 23 : 374 – 86 . Google Scholar CrossRef Search ADS PubMed 8 von Delius S , Schorn A , Grimm M , Schneider A , Wilhelm D , Schuster T et al. . Natural-orifice transluminal endoscopic surgery: low-pressure pneumoperitoneum is sufficient and is associated with an improved cardiopulmonary response (PressurePig Study) . Endoscopy 2011 ; 43 : 808 – 15 . Google Scholar CrossRef Search ADS PubMed 9 Higashi S , Nakajima K , Miyazaki Y , Makino T , Takahashi T , Kurokawa Y et al. . A case of transvaginal NOTES partial gastrectomy using new techniques and devices . Surg Case Rep 2015 ; 1 : 96. Google Scholar CrossRef Search ADS PubMed 10 Nakajima K , Takahashi T , Souma Y , Shinzaki S , Yamada T , Yoshio T et al. . Transvaginal endoscopic partial gastrectomy in porcine models: the role of an extra endoscope for gastric control . Surg Endosc 2008 ; 22 : 2733 – 6 . Google Scholar CrossRef Search ADS PubMed 11 Nakajima K , Nishida T , Takahashi T , Souma Y , Hara J , Yamada T et al. . Partial gastrectomy using natural orifice transluminal endoscopic surgery (NOTES) for gastric submucosal tumors: early experience in humans . Surg Endosc 2009 ; 23 : 2650 – 5 . Google Scholar CrossRef Search ADS PubMed 12 Kraitchman DL , Heldman AW , Atalar E , Amado LC , Martin BJ , Pittenger MF et al. . In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction . Circulation 2003 ; 107 : 2290 – 3 . Google Scholar CrossRef Search ADS PubMed 13 Miyagawa S , Sawa Y , Sakakida S , Taketani S , Kondoh H , Memon IA et al. . Tissue cardiomyoplasty using bioengineered contractile cardiomyocyte sheets to repair damaged myocardium: their integration with recipient myocardium . Transplantation 2005 ; 80 : 1586 – 95 . Google Scholar CrossRef Search ADS PubMed 14 Shimizu T , Sekine H , Yang J , Isoi Y , Yamato M , Kikuchi A et al. . Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues . FASEB J 2006 ; 20 : 708 – 10 . Google Scholar CrossRef Search ADS PubMed 15 Neudecker J , Sauerland S , Neugebauer E , Bergamaschi R , Bonjer HJ , Cuschieri A et al. . The European Association for Endoscopic Surgery clinical practice guideline on the pneumoperitoneum for laparoscopic surgery . Surg Endosc 2002 ; 16 : 1121 – 43 . Google Scholar CrossRef Search ADS PubMed 16 MacArthur JW , Goldstone AB , Cohen JE , Hiesinger W , Woo YJ. Cell transplantation in heart failure: where do we stand in 2016? Eur J Cardiothorac Surg 2016 ; 50 : 396 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Interactive CardioVascular and Thoracic SurgeryOxford University Press

Published: Jan 18, 2018

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