Keiichi Kanda

Mechanical stress-induced orientation and ultrastructural change of smooth muscle cells cultured in three-dimensional collagen lattices.

The effect of tensile stress on the orientation and phenotype of arterial smooth muscle cells (SMCs) cultured in three-dimensional (3D) type I collagen gels was morphologically investigated. Ring-shaped hybrid tissues were prepared by thermal gelation of a cold mixed solution of type I collagen and SMCs derived from bovine aorta. The tissues were subjected to three different modes of tensile stress. They were floated (isotonic control), stretched isometrically (static stress) and periodically stretched and recoiled by 5% above and below the resting tissue length at 60 RPM frequency (dynamic stress). After incubation for up to four wk, the tissues were investigated under a light microscope (LM) and a transmission electron microscope (TEM). Hematoxylin and eosinstained LM samples revealed that, irrespective of static or dynamic stress loading, SMCs in stress-loaded tissues exhibited elongated bipolar spindle shape and were regularly oriented parallel to the direction of the strain, whereas those in isotonic control tissues were polygonal or spherical and had no preferential orientation. In Azan-stained samples, collagen fiber bundles in isotonic control tissues were somewhat retracted around the polygonal SMCs to form a random network. On the other hand, those in statically and dynamically stressed tissues were accumulated and prominently oriented parallel to the stretch direction. Ultrastructural investigation using a TEM showed that SMCs in control and statically stressed tissues were almost totally filled with synthetic organelles such as rough endoplasmic reticulums, free ribosomes, Golgi complexes and mitochondria, indicating that the cells remained in the synthetic phenotype. On the other hand, SMCs in dynamically stressed tissues had increased fractions of contractile apparatus, such as myofilaments, dense bodies and extracellular filamentous materials equivalent to basement membranes, that progressed with incubation time. These results indicate that periodic stretch, in concert with 3-D extracellular collagen matrices, play a significant role in the phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular and biomolecular orientation.

Behavior of arterial wall cells cultured on periodically stretched substrates.

Cells in vital tissues align to form the most efficient configuration for functioning. Vascular cells of arterial walls are constantly exposed to fluid shear stress and pressure-induced periodic strain component, both of which are induced by pulsatile flow. In the present study, the effect of cyclic strain on the cellular orientation response and morphological changes of bovine arterial wall cells such as endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts (FCs) was studied. Cells seeded onto transparent elastomeric films were subjected to periodic stretch-relaxation under various amplitudes ranging from 5 to 20% and at frequencies ranging from 15 to 120 RPM for up to 24 h. Time-lapse video-recorded images of stress-loaded cells were analyzed by a computer-aided morphometric system to quantitatively evaluate the cellular orientation responses and morphological changes. The stress-loaded cells tended to align perpendicularly to the direction of stretch with time, regardless of cellular species. More pronounced orientation was attained under operating conditions with higher amplitude and frequency of stretching. The response of SMCs and FCs advanced more rapidly than that of ECs. Meanwhile, little morphological change was observed, irrespective of stress-loading or nonloading. Understanding of mechanically induced orientation response provides a fundamental basis on tissue engineering and biomechanics.

Differentiation from embryonic stem cells to vascular wall cells under in vitro pulsatile flow loading

This study evaluated the possibility of differentiation from embryonic stem (ES) cells to vascular wall cells by physical (mechanical) stress loading in vitro. A cell mixture containing Flk1-positive cells (ca. 30%) derived from murine ES cells was added to a compliant microporous tube made of segmented polyurethane. The compliance of the tube was close to that of the human artery [the stiffness parameter (β) = 57.2 (n = 5, SD < 5%)]. The luminal surface of the tube was fully covered with the cells by preincubation for two days in the presence of vascular endothelial growth factor (VEGF). After 2 days of additional incubation without VEGF under static conditions, layering of the grown cells, mostly smooth muscle actin (SMA)-positive cells, was observed only on the luminal surface of the tube. The cells were flat, polygonal, and randomly oriented. On the other hand, after a 2-day incubation under a weak pulsatile flow simulating the human venous systems [wall shear stress (WSS) from −0.98 to 2.2 dyn/cm2; circumferential strain (CS) 4.6–9.6 × 104 dyn/cm2] without VEGF, cells in the superficial layer were regularly oriented in the direction of the pulsatial flow. The oriented cells exhibited endothelial-like appearance, indicating that they were platelet endothelial cell adhesion molecule 1 (PECAM1)-positive. In addition, the cells growing into the interstices in the deeper layer showed smooth muscle-like appearance, indicating that they were SMA-positive. Differentiation to two different cell types and segregation of incorporated ES cells may be simultaneously encouraged by the combination of WSS and CS. It is expected that the monobloc building of hierarchically structured hybrid vascular prostheses composed of several vascular wall cell types is possible by physically synchronized differentiation of ES cells.

Highly oriented, tubular hybrid vascular tissue for a low pressure circulatory system.

A hierarchically structured hybrid vascular tissue was prepared from vascular cells and collagen. First a hybrid medial tissue was prepared by pouring a cold mixed solution of bovine aortic smooth muscle cells (SMCs) and Type I collagen into a tubular glass mold composed of a mandrel and a sheath (inner diameter, 1.5 mm; outer diameter, 7 mm; length, 7 cm). An SMC incorporating collagenous gel was formed with incubation at 37 degrees C. After the sheath of the mold was removed, the resulting fragile tissue cultured in the medium shrank in a time dependent manner to form an opaque, dense tissue. Seeding at a higher cell density and a lower concentration of collagen resulted in rapid and prominent shrinkage. Morphologic investigation showed that with time, bipolarly elongated SMCs and collagen fiber bundles became positioned around the mandrel. When the mandrel was removed, a tubular hybrid medial tissue was formed. A hybrid vascular tissue with a hierarchical structure was constructed by seeding endothelial cells onto the inner surface of the hybrid medial tissue. Prepared tissues tolerated luminal pressures as great as 100 mmHg and mechanical stress applied during the anastomotic procedure. This method allowed the authors to prepare hybrid medial tissues of predetermined size (specifically inner diameter, wall thickness, and length) and mechanical property, which all depend on the mold design, SMC seeding density, initial collagen concentration, and incubation period. Hybrid vascular tissues may provide physiologic functions such as antithrombogenicity and regulation of vasomotor tone when implanted into a venous system.

Mechanical stress induced cellular orientation and phenotypic modulation of 3-D cultured smooth muscle cells.

The effect of periodic stretch on cellular orientation and intracellular ultrastructure of three-dimensionally (3-D) cultured arterial smooth muscle cells (SMCs) was investigated. Ring shaped hybrid tissues were prepared by thermal gelation of a mixed solution of Type I collagen and SMCs derived from bovine aorta. The gels were subjected to three modes of stress loading: floated (control), stretched isometrically (static stress), and periodically stretched and recoiled with 10% amplitude at 60 rpm frequency (dynamic stress). After 4 weeks of stress loading, the gels were morphologically investigated with a light microscope and a transmission electron microscope (TEM). Irrespective of static or dynamic stress loading, SMCs in stress loaded gels exhibited an elongated bipolar spindle shape and were oriented parallel to the direction of stretch, whereas those in control gels were polygonal shaped and randomly oriented. TEM observation showed that SMCs in control and static stress loaded gels were intracellularly filled with organelles, such as rough endoplasmic reticulum, free ribosomes, Golgi complexes, and mitochondria, indicating that the cells were of the synthetic phenotype. On the other hand, SMCs in dynamic stress loaded gels tended to have increased contractile apparatus, such as myofilaments, dense bodies, and basement membranes, suggesting that periodic stretch plays an important role in phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular orientation.

In vitro reconstruction of hybrid vascular tissue : hierarchic and oriented cell layers

Hybrid vascular tissue was hierarchically reconstructed in vitro. A hybrid medial layer composed of type I collagen gel, in which SMCs derived from a mongrel dog were embedded, was formed on the inner surface of a compliant porous polyurethane graft (internal diameter = 3 mm). Endothelial cells (ECs) from the same animal were seeded and cultured on the hybrid media to build an intimal layer. Subsequently, hierarchically structured grafts constructed in this manner were subjected to pulsatile flow (flow rate: 8.5 ml/min; frequency: 60 rpm; amplitude: 5% of graft outer diameter) of culture medium (Medium 199 supplemented with 20% fetal calf serum). After stress loading for as long as 10 days, tissues were morphologically investigated with a light microscope and a scanning electron microscope. Inner surfaces of the hybrid tissues were covered with EC monolayers that aligned along the direction of the flow (i.e., longitudinally). However, SMCs beneath the intima aligned in the circumferential direction. These cellular orientations resembled those in native muscular arteries. The pulsatile stress loaded hybrid tissue mimicked native muscular arteries with respect to hierarchic structure and cellular orientation. In vitro mechanical stress loading on a hybrid graft might provide a high degree of integrity in terms of tissue structure that promises high tolerance toward hydrodynamic stress and regulation of vasomotor tone upon implantation.

Development of a Completely Autologous Valved Conduit With the Sinus of Valsalva Using In-Body Tissue Architecture Technology A Pilot Study in Pulmonary Valve Replacement in a Beagle Model

Background— We developed autologous prosthetic implants by simple and safe in-body tissue architecture technology. We present the first report on the development of autologous valved conduit with the sinus of Valsalva (BIOVALVE) by using this unique technology and its subsequent implantation in the pulmonary valves in a beagle model. Methods and Results— A mold of BIOVALVE organization was assembled using 2 types of specially designed silicone rods with a small aperture in a trileaflet shape between them. The concave rods had 3 projections that resembled the protrusions of the sinus of Valsalva. The molds were placed in the dorsal subcutaneous spaces of beagle dogs for 4 weeks. The molds were covered with autologous connective tissues. BIOVALVEs with 3 leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds. These valves had adequate burst strength, similar to that of native valves. Tight valvular coaptation and sufficient open orifice area were observed in vitro. These BIOVALVEs were implanted to the main pulmonary arteries as allogenic conduit valves (n=3). Postoperative echocardiography demonstrated smooth movement of the leaflets with trivial regurgitation. Histological examination of specimens obtained at 84 days showed that the surface of the leaflet was covered by endothelial cells and neointima, including an elastin fiber network, and was formed at the anastomosis sides on the luminal surface of the conduit. Conclusion— We developed the first completely autologous BIOVALVE and successfully implanted these BIOVALVEs in a beagle model in a pilot study.

In vitro reconstruction of hybrid arterial media with molecular and cellular orientations.

A hybrid medial tissue composed of a type I collagen gel, into which smooth muscle cells (SMCs) derived from bovine aortic media were 3-dimensionally (3D) embedded, was constructed around an elastomeric silicone tube (outer diameter: 8 mm). Subsequently, hybrid tissues thus prepared were subjected to three modes of mechanical stimulation in the medium: one was subjected to flotation with no disturbance (isotonic control), the second was kept isometrically (static stress) and the third was subjected to continuous periodic stretch by inflation of the embedded silicone tube which simulated arterial pulsation (dynamic stress, amplitude: 5% in inner diameter; frequency: 60 RPM). After a 5-day culture period, hybrid tissues were morphologically investigated. In control gels, polygonal SMCs and extracellular collagen fiber bundles were randomly oriented. On the other hand, upon static or dynamic stress loading, bipolar spindle-shaped SMCs and dense collagen fiber bundles were aligned circumferentially around the silicone tube, which proceeded with time. The orientations of SMCs and collagen fibers were more prominent in dynamically stressed hybrid tissues than those in statically stressed ones. The pulsatile stress-loaded hybrid medial tissue mimicked the media of native muscular arteries in terms of cellular and molecular orientations.

Local elasticity imaging of vascular tissues using a tactile mapping system

This study aimed to map the elasticity of a natural artery at the micron level by using a tactile mapping system (TMS) that was recently developed for characterization of the stiffness of tissue slices. The sample used was a circumferential section (thickness, approximately 1 mm) of a small-caliber porcine artery (diameter, approximately 3 mm). Elasticity was measured with a probe of diameter 1 μm and a spatial resolution of 2 μm at a rate of 0.3 s per point, without significant sample invasion. Topographical measurements were also performed simultaneously. Wavy regions of high elasticity, layered in the circumferential direction, were measured at the tunica media, which was identified as an elastin-rich region. The Young’s modulus of the elastin-rich region in the media was 50.8 ± 13.8 kPa, and that of the elastin-rich region of the lamina elastica interna was 69.0 ± 12.8 kPa. Both these values were higher than the Young’s modulus of the other regions in the media, including smooth muscle cells and collagen fibrils (17.0 ± 9.0 kPa). TMS is simple and inexpensive to perform and allows observation of the distribution of the surface elastic modulus at the extracellular matrix level in vascular tissue. TMS is expected to be a powerful tool in evaluation of the maturation and degree of reconstruction in the development of tissue-engineered or artificial tissues and organs.

In-body tissue-engineered aortic valve (Biovalve type VII) architecture based on 3D printer molding

In-body tissue architecture--a novel and practical regeneration medicine technology--can be used to prepare a completely autologous heart valve, based on the shape of a mold. In this study, a three-dimensional (3D) printer was used to produce the molds. A 3D printer can easily reproduce the 3D-shape and size of native heart valves within several processing hours. For a tri-leaflet, valved conduit with a sinus of Valsalva (Biovalve type VII), the mold was assembled using two conduit parts and three sinus parts produced by the 3D printer. Biovalves were generated from completely autologous connective tissue, containing collagen and fibroblasts, within 2 months following the subcutaneous embedding of the molds (success rate, 27/30). In vitro evaluation, using a pulsatile circulation circuit, showed excellent valvular function with a durability of at least 10 days. Interposed between two expanded polytetrafluoroethylene grafts, the Biovalves (N = 3) were implanted in goats through an apico-aortic bypass procedure. Postoperative echocardiography showed smooth movement of the leaflets with minimal regurgitation under systemic circulation. After 1 month of implantation, smooth white leaflets were observed with minimal thrombus formation. Functional, autologous, 3D-shaped heart valves with clinical application potential were formed following in-body embedding of specially designed molds that were created within several hours by 3D printer.