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Teruo Okano

Publications and source records attributed to Teruo Okano.

At least 37 records · Page 2Linked to original sources

Study of temperature-responsibility on the surfaces of a thermo-responsive polymer modified stationary phase.

We investigated a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which is the basis of an HPLC stationary phase. We prepared a PNIPAAm terminally-modified surface. In this study, we investigated the effect of PNIPAAm on the surface of a stationary phase on separation based on changes of the retention time with the temperature step gradient. As the temperature changed the surface property of the stationary phase switched from hydrophilic to hydrophobic. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. Using a column packed with PNIPAAm-modified silica, the separation of steroids was carried out by changing the temperature. With increasing temperature, an increased interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases was observed. A temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The PNIPAAm-modified surface of the stationary phase exhibited temperature-controlled hydrophilic-hydrophobic changes. The drastic and reversible surface hydrophilic-hydrophobic property alteration for PNIPAAm terminally-grafted surfaces should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. This system should be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent.

Acrylic Resins↗

Pulsatile cardiac tissue grafts using a novel three-dimensional cell sheet manipulation technique functionally integrates with the host heart, in vivo.

We devised a method of fabricating easily transplantable scaffoldless 3D heart tissue, made with a novel cell-sheet (CS) technology from cultured cardiomyocytes using a fibrin polymer coated dish. In the present study, we tested in vivo electrical communication which is essential for improving heart function between the host heart and the grafted CS. The epicardial surface of the ventricle of an anesthetized open-chest nude rat was ablated by applying a heated metal. Bilayered CS was obtained from neonatal rat primary culture. CS was transplanted onto the injured myocardial surface (sMI) (sMI+sheet group). The rats were allowed to recover for 1 to 4 weeks, to stabilize the grafts. Action potentials (APs) from the excised perfused heart were monitored by the fluorescence signal of di-4ANEPPS with a high speed charge-coupled device camera. The APs were observed under epicardial pacing of the host heart or the CS grafts. The pacing threshold of the current output was measured in the sMI+sheet group and in the nongrafted sMI group at the center of the sMI and in the normal zone (Nz). Bidirectional AP propagation between the sMI and Nz was observed in the sMI+sheet group (n=14), but was blocked at the marginal area of the sMI in the sMI group (n=9). The ratio of the pacing threshold (sMI/Nz) was significantly lower in the sMI+sheet than in the sMI group (3.0+/-0.7, 19.0+/-6.1 respectively P<0.05). There were neither spontaneous nor pacing-induced arrhythmias in these two groups. Bidirectional smooth AP propagation between the host heart and the grafted CS was observed. This finding suggested functional integration of this CS graft with the host heart without serious arrhythmia.

Action Potentials↗

Aqueous chromatography system using pH- and temperature-responsive stationary phase with ion-exchange groups.

We report on the development of a novel analytical HPLC technique of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, ketoprofen and naproxen, with an isocratic aqueous mobile phase. In this study, we designed a new pH- and temperature-responsive copolymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm). The copolymer was modified with cross-linked poly(NIPAAm-co-BMA-co-DMAPAAm) (IBD) hydrogel on to aminopropyl silica beads, and the products were evaluated as HPLC packing materials for an ion-exchange- and temperature-responsive chromatography. The property of the surface of the stationary phase was altered from hydrophilic to hydrophobic, and from charged to non-charged by changes in the temperature and pH. In addition, it is possible that ion-exchange groups can appear or be hidden on the polymer chain surface by temperature changes. The interactions of NSAIDs with this stationary phase were controlled by the temperature and the pH with a constant aqueous mobile phase. PH- and temperature-responsive chromatography is expected to be useful for the separation of pharmaceuticals and biomolecules.

Acrylamides↗

Preparation of camptothecin-loaded polymeric micelles and evaluation of their incorporation and circulation stability.

To improve its aqueous solubility and stability in biological fluid, CPT was physically loaded in polymeric micelles. Polymeric micelles were composed of various poly(ethylene glycol)-poly(aspartate ester) block copolymers (PEG-P(Asp(R))). The incorporation and circulation stability of CPT micelles were evaluated by measuring the CPT in micelle using gel-permeation chromatography and by CPT concentration measurement after intravenous injection using HPLC, respectively, in terms of chemical structure of block copolymers. The stability of CPT-loaded micelles in vivo depended on the amount of benzyl esters, and length of PEG in the polymers to a greater degree than it did in vitro. A stable formulation of CPT-loaded micelles was obtained using PEG-P(Asp) with PEG of 5,000 (MW), 27 Asp units, and 57-75% benzyl esterification of Asp residue. This CPT-loaded micelles showed about a 17-fold lower blood clearance value than unstable micelles. The CPT-loaded micelles are potentially delivered to tumor sites owing to an extended circulation in the blood stream.

Animals↗

Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters.

OBJECTIVES: Cell therapy is a promising strategy for ischemic cardiomyopathy. However, the direct injection method has limitations for generalized cell delivery, especially in dilated cardiomyopathy (DCM). We hypothesized that a sheet-shaped myoblast graft would be superior to direct injection for improving cardiac performance in DCM. METHODS: Male 27-week-old BIO TO-2 (DCM) hamsters that showed moderate cardiac remodeling were used as recipients. Myoblasts isolated from BIO F1B hamsters were cultured on dishes coated with poly(N-isopropylacrylamide), a temperature-responsive polymer, and spontaneously detached as a sheet-shaped graft at 20 degrees C without enzymatic treatment. Three different therapies were conducted: (1) sheet-shaped myoblast graft implantation (S group, n=29); (2) myoblast injection (M group, n=28); and (3) sham operation (C group, n=28). In the S group, two sheet-shaped myoblast grafts were implanted on the left ventricle (LV) wall, and in the M group, myoblasts were injected into the right ventricle (RV) and LV walls. RESULTS: After the sheet-shaped myoblast grafts were implanted, echocardiography demonstrated that the dilated LV dimension was significantly reduced, whereas the hearts in other groups showed a progression of LV dilation. The fractional shortening in the M and C groups decreased significantly while that in the S group was maintained at the preoperative level for 3 months after the operation. Histological examination demonstrated that in the S group, the LV wall thickness was increased, with viable myoblasts, and myocardial fibrosis was decreased compared with the other groups. Immunohistochemical staining demonstrated alpha-sarcoglycan and beta-sarcoglycan expression on the basement membrane of the cardiomyocytes in the S group but not in the other groups. The life expectancy was significantly prolonged in the S group. CONCLUSION: Sheet-shaped myoblast graft implantation improved cardiac performance and prolonged life expectancy, associated with a reduction in myocardial fibrosis and re-organization of the cytoskeletal proteins in DCM hamsters. Thus, sheet-shaped autologous myoblast graft implantation may induce restoration of the heart in DCM.

Animals↗

Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.

Recently, the field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Therefore, new technologies for fabricating functional tissues with a well-organized vasculature are required. In the present study, neonatal rat cardiomyocytes were harvested as intact sheets from temperature-responsive culture dishes and stacked into cell-dense myocardial tissues. However, the thickness limit for layered cell sheets in subcutaneous tissue was approximately 80 microm (3 layers). To overcome this limitation, repeated transplantation of triple-layer grafts was performed at 1, 2, or 3 day intervals. The two overlaid grafts completely synchronized and the whole tissues survived without necrosis in the 1 or 2 day interval cases. Multistep transplantation also created approximately 1 mm thick myocardium with a well-organized microvascular network. Furthermore, functional multilayer grafts fabricated over a surgically connectable artery and vein revealed complete graft perfusion via the vessels and ectopic transplantation of the grafts was successfully performed using direct vessel anastomoses. These cultured cell sheet integration methods overcome long-standing barriers to producing thick, vascularized tissues, revealing a possible solution for the clinical repair of various damaged organs, including the impaired myocardium.

Animals↗

An actuated pump on-chip powered by cultured cardiomyocytes.

Cellular functions are frequently exploited as processing components for integrated chemical systems such as biochemical reactors and bioassay systems. Here, we have created a new cell-based microsystem exploiting the intrinsic pulsatile mechanical functions of cardiomyocytes to build a cellular micropump on-chip using cardiomyocyte sheets as prototype bio-microactuators. We first demonstrate cell-based control of fluid motion in a model microchannel without check valves and evaluate the potential performance of the bio-actuation. For this purpose, a poly(dimethylsiloxane) (PDMS) microchip with a microchannel equipped with a diaphragm and a push-bar structure capable of harnessing collective cell fluid mechanical forces was coupled to a cultured pulsating cardiomyocyte sheet, activating cell-based fluid movement in the microchannel by actuating the diaphragm. Cell oscillation frequency and correlated fluid displacement in this system depended on temperature. When culture temperature was increased, collective cell contraction frequency remained cooperative and synchronous but increased, while displacement was slightly reduced. We then demonstrated directional fluid pumping within microchannels using cantilever-type micro-check valves made of polyimide. A directional flow rate of nL min(-1) was produced. This cell micropump system could be further developed as a self-actuated and efficient mechanochemical transducer requiring no external energy sources for various purposes in the future.

Animals↗

Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential.

Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive culture dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.

Animals↗

Demonstration of a PDMS-based bio-microactuator using cultured cardiomyocytes to drive polymer micropillars.

Natural cellular functions are increasingly exploited for integrated chemical systems such as biochemical reactors and biosensors. We propose to utilize the intrinsic mechanical function of cardiomyocytes, converting chemical energy into mechanical energy. In this report, we demonstrate the working principle of our proposed poly(dimethylsiloxane) (PDMS) based cardiomyocyte bio-microactuator using fabricated PDMS micropillars driven to repetitive motion by attached pulsating cardiomyocytes. Sheets of PDMS embedded with an array of micropillars were fabricated and modified for cardiomyocyte attachment in culture. Primary neonatal rat cardiomyocytes were cultured on the array, attaching to the micropillars and substratum successfully, and exhibiting their typical spontaneous, pulsatile phenotype. Micropillars beat with the coupled cells spontaneously without any triggers. The beat frequency was 1.4 Hz at 37 degrees C and the displacement of the top of the pillar that beat most strongly in our observation was 2.8+/-0.2 microm. From this result, contractile forces of cultured cardiomyocytes were estimated to exceed 3.5 microN. The estimated force is far greater than that of a previously described hydrogel-based cardiomyocyte bio-microactuator (K. Morishima et al., in Micro Total Analysis Systems 2003, ed. M. A. Northrup et al., The Transducers Research Foundation, San Diego, CA, vol. 2, pp. 1125-1128). PDMS compatibility as a base material for bio-microactuator design using cultured cardiomyocytes was verified. This PDMS-based cell microactuator worked for about one week without exchange of the culture medium, and this system could be developed for various purposes in the future as self-actuated and efficient mechanochemical transducers without external energy source requirements.

Algorithms↗

Cardiomyocyte bridging between hearts and bioengineered myocardial tissues with mesenchymal transition of mesothelial cells.

BACKGROUND: For the reconstruction of 3-dimensional (3D) tissues, we exploited an original method of tissue engineering that layers individual cell sheets harvested from temperature-responsive culture dishes. Stacked cardiomyocyte sheets demonstrated electrical and morphologic communication, resulting in synchronously beating myocardial tissue. When these bioengineered 3D tissue grafts are transplanted onto damaged hearts, gap junction communication between graft and host is likely critical for synchronized beating and functional improvement. In this study, these graft-to-heart morphologic communications were examined. METHODS: Neonatal rat cardiomyocyte sheets were harvested from temperature-responsive culture dishes and layered to create 3D tissues. These constructs were then transplanted onto infarcted rat hearts. Histologic analyses and transmission electron microscopy (TEM) were performed to examine morphologic communications. The passage of small molecules through functional gap junctions was also detected using a dye-transfer assay. RESULTS: Transplanted cardiomyocytes bridged between the grafts and hearts in intact areas. Connexin-43 staining and TEM revealed the existence of gap junctions and intercalated disks between the bridging cardiomyocytes. Furthermore, it was confirmed that a low-molecule fluorescent dye, calcein, was transferred from the grafts to the hearts via the bridging cardiomyocytes. Immunohistochemistry with anti-intercellular adhesion molecule-1 antibodies revealed that mesothelial cells in the epicardium scattered and transdifferentiated into mesenchymal cells between the graft and host. CONCLUSIONS: The direct attachment of layered cardiomyocyte sheets on the heart surface promotes mesothelial cell transdifferentiation and cardiomyocyte bridging, leading to functional communication via gap junctions. These results indicate that these bioengineered myocardial tissues may improve damaged heart function via synchronized beating.

Animals↗

Effects of carbocisteine on sialyl-Lewis x expression in an airway carcinoma cell line stimulated with tumor necrosis factor-alpha.

Carbocisteine is a mucoregulatory drug normalizing sialic acid and fucose contents in mucins through the regulation of glycosyltransferase activities. Tumor necrosis factor (TNF)-alpha-induced overexpression of sialyl-Lewis x epitopes, containing sialic acid and fucose, in mucins were previously reported to be regulated by glycosyltransferase mRNAs expression through phosphatidyl inositol-specific phospholipase C (PI-PLC) signaling pathways [Ishibashi, Y., Inouye, Y., Okano, T., Taniguchi, A., 2005. Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line. Glycoconj. J. 22, 53-62]. To investigate the mechanism behind the mucoregulatory action of carbocisteine, the present study evaluated the effects of carbocisteine on TNF-alpha-induced overexpression of sialyl-Lewis x epitopes in NCI-H292 cells. 100 mug/ml of carbocisteine was able to inhibit the TNF-alpha-induced expression of hST3GallV mRNA, FUT3 mRNA, C2/4GnT mRNA and sialyl-Lewis x epitopes as well as the TNF-alpha-induced activity of PI-PLC in NCI-H292 cells. These findings suggest that carbocisteine may normalize the sialyl-Lewis x epitopes expression in mucins through the inhibition of cellular PI-PLC activity in vivo.

Carbocysteine↗

Thermal modulated interaction of aqueous steroids using polymer-grafted capillaries.

Poly(N-isopropylacrylamide) (PIPAAm) of controlled molecular weight was densely grafted onto glass capillary lumenal surfaces using surface-initiated atom transfer radical polymerization (ATRP). Temperature-dependent changes of these thermoresponsive brush surfaces with hydrophobic steroids were investigated by exploiting thermoresponsive aqueous wettability changes of the polymer-modified surfaces in microfluidic systems. IPAAm was polymerized on ATRP initiator-immobilized glass surfaces using CuCl/CuCl(2)/tris(dimethylaminoethyl)amine (Me(6)TREN) as an ATRP catalyst in water at 25 degrees C. PIPAAm graft layer thickness and its homogeneity on glass surfaces are controlled by changing ATRP reaction time. Aqueous wettability changes of PIPAAm-grafted surfaces responses drastically changed to both grafted polymer layer thickness and temperature, especially at lower temperatures. Temperature-responsive surface properties of these PIPAAm brushes within capillary inner wall surfaces were then investigated using capillary chromatography. Effective interaction of hydrophobic steroids with dehydrated, hydrophobized PIPAAm-grafted capillary surfaces was observed above 30 degrees C without any column packing materials. Steroid elution behavior from PIPAAm-grafted capillaries contrasted sharply with that from PIPAAm hydrogel-grafted porous monolithic silica capillaries prepared by electron beam (EB) irradiation wherein significant peak broadening was observed at high-temperature regardless of sample hydrophobicity factors (log P values), indicating multistep separation modes in coated monolithic silica capillaries. In conclusion, thermoresponsive polymer-grafted capillary inner wall surfaces prepared by ATRP exhibit useful temperature-dependent surface property alterations effective to regulate interactions with biomolecules without requirements for separation bed packing materials within the capillary lumen.

Journal Article↗

Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells.

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.

Animals↗

Maskless liquid-crystal-display projection photolithography for improved design flexibility of cellular micropatterns.

We previously developed an all-in-one photopolymerization device by modifying a commercially available liquid crystal display projector (LCDP) for the preparation of micropatterned surfaces and microfluidic channels without the need for expensive photomasks. In the present study, we demonstrate a simple and reliable method for rapid prototyping of cell micropatterning with high resolution using the modified LCDP device. Fabrication of complicated and flexible patterns was achieved using this device with positive-type photoresist in a two-step process. First, micropatterns on the silanized coverslips were fabricated from positive photoresist. Second, acrylamide monomer solution containing polymerization initiator was dropped onto the micropatterned positive photoresist and copolymerized on the silanized coverslips in situ by thermally initiated radical polymerization. After the reaction, the remnant micropatterned photoresist is easily dissolved, resulting in a polyacrylamide-silane micropattern on the coverslip. The resultant polyacrylamide layer is highly hydrophilic and repels both protein adsorption and cell adhesion. Cells seeded on the micropatterned surfaces therefore attach and spread only on unpolymerized silanized glass surfaces, conforming to the pattern design. This technique is therefore useful for inexpensive, rapid prototyping of surface micropatterns using polymer materials.

Acrylic Resins↗

Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-induced canine heart failure model.

OBJECTIVE: To overcome problems related to the intramyocardial injection of cells, including cell loss and a limited graft area, we developed a cell delivery system that uses tissue-engineered myoblast grafts grown as sheets. Here, we assessed the feasibility and efficacy of our method in a canine dilated cardiomyopathy model. METHODS: Skeletal myoblasts were incubated on temperature-responsive culture dishes, and the sheets of cells were detached by decreasing the temperature. Twelve dogs were given continuous ventricular pacing at 230 beats/min for 4 weeks; then the myoblast sheets (n = 5) were grafted onto the left ventricular wall or a sham operation was performed (n = 7). The number of cells was adjusted to 1.5 approximately 2.5 x 10(6) cells per graft, and each dog received approximately 20 grafts. RESULTS: The cell sheets were easily grafted onto a large area of the left ventricular surface, and there were no serious sequelae. Four weeks after graft implantation, echocardiography demonstrated that the left ventricular ejection fraction (graft, 26.0% +/- 5.6%; control, 19.5% +/- 6.8%; P < .05) and fractional shortening (graft, 17.9% +/- 3.6%; control, 7.8% +/- 2.1%; P < .05) were significantly ameliorated with reduced left ventricular dilatation (graft, 7.3 +/- 1.3 cm2; control, 10.2 +/- 0.4 cm2; P < .05) and increased wall thickness (graft, 5.6 +/- 0.7 mm; control, 4.4 +/- 0.6 mm; P < .05). Histologic evidence indicated the grafted myoblasts had survived, accompanied by a significant reduction in fibrosis and apoptosis, and a significant increase in proliferation. CONCLUSIONS: Grafting of skeletal myoblast sheets attenuated cardiac remodeling and improved cardiac performance. This novel method was feasible and effective in a large animal model, suggesting an innovative and promising strategy for treating patients with end-stage dilated cardiomyopathy.

Animals↗

Corneal epithelial stem cell delivery using cell sheet engineering: not lost in transplantation.

Cell-based therapies have now generated significant interest as novel drug delivery systems, with various adult cell types used in treating a wide range of diseases. To overcome the limits that restrict treatments for corneal surface dysfunction, corneal epithelial stem cells expanded ex vivo have been applied as an alternative approach. While previous studies used various carrier substrates, we present a novel method using cell sheet engineering with temperature-responsive culture dishes to create carrier-free corneal epithelial stem cell sheets that can be transplanted without sutures. Results from clinical trials reveal successful transplantation with the recovery of lost visual acuity in all cases. Cell sheet engineering, therefore, presents a novel method for the delivery of corneal epithelial stem cells, and can also be applied for other approaches of cellular therapeutics.

Corneal Transplantation↗

Tissue engineered epithelial cell sheets for the creation of a bioartificial trachea.

To successfully engineer a bioartificial tracheal replacement, it is believed that the regeneration of a functional epithelial lining is a key requirement. In the present study, rabbit tracheal epithelial cells were cultured on temperature-responsive culture dishes, under normal culture conditions at 37 degrees C. By simple temperature reduction to 20 degrees C, the cultured epithelial cells were noninvasively harvested as intact sheets, without the use of any proteolytic enzymes. Support Dacron grafts that had been subcutaneously implanted for 4 weeks to allow for host tissue and vessel infiltration were then opened, and the tracheal epithelial cell sheets were transplanted to the luminal surface without sutures. These fabricated constructs were then used as tracheal replacements, in a rabbit model. Four weeks after transplantation, results showed that the tracheal grafts were covered by a mature, pseudostratified columnar epithelium. In contrast, control constructs that did not receive cell sheet transplantation demonstrated only a thin, immature epithelium at the center of the replacement graft. These results therefore demonstrate that these tracheal epithelial cell sheets can create an epithelial lining on the luminal surface of a bioartificial trachea.

Animals↗

Transplantation of an autologous mesothelial cell sheet prepared from tunica vaginalis prevents post-operative adhesions in a canine model.

Post-operative adhesions often cause severe complications such as bowel obstruction and abdominopelvic pain. Previously, we reported that transplantation of a mesothelial cell sheet is effective for preventing adhesion in rat model. We also proposed a new technique for harvesting autologous mesothelial cells from tunica vaginalis without intra-abdominal maneuvers. In this study, we examined whether an autologous mesothelial cell sheet can prevent post-operative peritoneal adhesions in a canine adhesion model. Mesothelial cells were isolated from the tunica vaginalis of male beagles. Isolated cells were cultured on fibrin gel. We named this construct the "mesothelial cell sheet." Animals underwent surgery to induce peritoneal adhesion formation and were then transplanted with the mesothelial cell sheets (sheet group, n = 4), fibrin gel (fibrin group, n = 4), or no materials (sham group, n = 4). Four weeks after the transplantation, we evaluated adhesion formation and scored adhesion levels. The abdominal wall transplanted with the mesothelial cell sheet was covered with mesothelium. The total adhesion score of the sheet group was significantly lower than that of the fibrin group and the sham group. These results indicated that transplantation of an autologous mesothelial cell sheet is effective for preventing post-operative adhesion formation in the canine adhesion model. Our mesothelial cell sheet has the potential to be a powerful adhesion prophylactic material in surgery.

Abdominal Wall↗