Securing high-quality cell sources is important in regenerative medicine. In this study, we developed a device that can accumulate autologous stem cells in the body. When small wire-assembled molds were embedded in the dorsal subcutaneous pouches of beagles for several weeks, collagen-based tissues with minimal inflammation formed inside the molds. At 3 weeks of embedding, the outer areas of the tissues were composed of immature type III collagen with large amounts of cells expressing SSEA3 or SSEA4 markers, in addition to growth factors such as HGF or VEGF. When separated from the tissues by collagenase treatment, approximately four million cells with a proportion of 70% CD90-positive and 20% SSEA3- or SSEA4-positive cells were recovered from the single mold. The cells could differentiate into bone or cartilage cells. The obtained cell-containing tissues are expected to have potential as therapeutic materials or cell sources in regenerative medicine.

There is a great clinical need for regenerating urinary tissue. Native urethras and ureters have bidirectional aligned smooth muscle cells (SMCs) layers, which plays a pivotal role in micturition and transporting urine and inhibiting reflux. Thus far, urinary scaffolds have not been designed to induce the native-mimicking aligned arrangement of SMCs. In this study, a tubular decellularized extracellular matrix (dECM) with an intact internal layer and bidirectional aligned microchannels in the tubular wall, which is realized by the subcutaneous implantation of a template, followed by the removal of the template, and decellularization, is engineered. The dense and intact internal layer effectively increases the leakage pressure of the tubular dECM scaffolds. Rat-derived dECM scaffolds with three different sizes of microchannels are fabricated by tailoring the fiber diameter of the templates. The rat-derived dECM scaffolds exhibiting microchannels of ≈65 µm show suitable mechanical properties, good ability to induce the bidirectional alignment and growth of human bladder SMCs, and elevated higher functional protein expression in vitro. These data indicate that rat-derived tubular dECM scaffolds manifesting double-layer aligned microchannels may be promising candidates to induce the native-mimicking regeneration of SMCs in urethra and ureter reconstruction.

Cell-based therapy and tissue engineering are promising substitutes for liver transplantation to cure end-stage liver disorders. However, the limited sources for healthy and functional cells and poor engraftment rate are main challenges to the cell-based therapy approach. On the other hand, feasibility of production and size of bioengineered tissues are primary bottlenecks in tissue engineering. Here, we induce regeneration in a rat fibrotic liver model by transplanting a natural bioengineered scaffold with a native microenvironment repopulated with autologous stem/progenitor cells. In the main experimental group, a 1 mm3 stromal derived factor-1α (SDF-1α; S) loaded scaffold from decellularized liver extracellular matrix (LEM) was transplanted (Tx) into a fibrotic liver and the endogenous stem/progenitor cells were mobilized via granulocyte colony stimulating factor (G-CSF; G) therapy. Four weeks after transplantation, changes in liver fibrosis and necrosis, efficacy of cell engraftment and differentiation, vasculogenesis, and liver function recovery were assessed in this (LEM-TxSG) group and compared to the other groups. We found significant reduction in liver fibrosis stage in the LEM-TxSG, LEM-TxS and LEM-TxG groups compared to the control (fibrotic) group. Liver necrosis grade, and alanine transaminase (ALT) and aspartate transaminase (AST) levels dramatically reduced in all experimental groups compared to the control group. However, the number of engrafted cells into the transplanted scaffold and ratio of albumin (Alb) positive cells per total incorporated cells were considerably higher in the LEM-TxSG group compared to the LEM-Tx, LEM-TxS and LEM-TxG groups. Serum Alb levels increased in the LEM-Tx, LEM-TxS, and LEM-TxG groups, and was highest in the LEM-TxSG group, which was significantly more than the fibrotic group. Small vessel formation in the LEM-TxSG group was significantly higher than the LEM-Tx and LEM-TxS groups. Totally, these findings support application of the in vivo tissue engineering approach as a possible novel therapeutic strategy for liver fibrosis.

Mechanical and bioprosthetic valves that are currently applied for replacing diseased heart valves are not fully efficient. Heart valve tissue engineering may solve the issues faced by the prosthetic valves in heart valve replacement. The leaflets of native heart valves have a trilayered structure with layer-specific orientations; thus, it is imperative to develop functional leaflet tissue constructs with a native trilayered, oriented structure. Its key solution is to develop leaflet scaffolds with a native morphology and structure. In this study, microfibrous leaflet scaffolds with a native trilayered and oriented structure were developed in an electrospinning system. The scaffolds were implanted for 3 months in rats subcutaneously to study the scaffold efficiencies in generating functional tissue-engineered leaflet constructs. These in vivo tissue-engineered leaflet constructs had a trilayered, oriented structure similar to native leaflets. The tensile properties of constructs indicated that they were able to endure the hydrodynamic load of the native heart valve. Collagen, glycosaminoglycans, and elastin─the predominant extracellular matrix components of native leaflets─were found sufficiently in the leaflet tissue constructs. The residing cells in the leaflet tissue constructs showed vimentin and α-smooth muscle actin expression, i.e., the constructs were in a growing state. Thus, the trilayered, oriented fibrous leaflet scaffolds produced in this study could be useful to develop heart valve scaffolds for successful heart valve replacements.

Tissue-engineered heart valves are a promising alternative solution to prosthetic valves. However, long-term functionalities of tissue-engineered heart valves depend on the ability to mimic the trilayered, oriented structure of native heart valve leaflets. In this study, using electrospinning, we developed trilayered microfibrous leaflet substrates with morphological characteristics similar to native leaflets. The substrates were implanted subcutaneously in rats to study the effect of their trilayered oriented structure on in vivo tissue engineering. The tissue constructs showed a well-defined structure, with a circumferentially oriented layer, a randomly oriented layer and a radially oriented layer. The extracellular matrix, produced during in vivo tissue engineering, consisted of collagen, glycosaminoglycans, and elastin, all major components of native leaflets. Moreover, the anisotropic tensile properties of the constructs were sufficient to bear the valvular physiological load. Finally, the expression of vimentin and α-smooth muscle actin, at the gene and protein level, was detected in the residing cells, revealing their growing state and their transdifferentiation to myofibroblasts. Our data support a critical role for the trilayered structure and anisotropic properties in functional leaflet tissue constructs, and indicate that the leaflet substrates have the potential for the development of valve scaffolds for heart valve replacements.

A tissue-engineered heart valve can be an alternative to a prosthetic valve in heart valve replacement; however, it is not fully efficient in terms of long-lasting functionality, as leaflets in engineered valves do not possess the trilayered native leaflet structure. Previously, we developed a flat, trilayered, oriented nanofibrous (TN) scaffold mimicking the trilayered structure and orientation of native heart valve leaflets. In vivo tissue engineering-a practical regenerative medicine technology-can be used to develop an autologous heart valve. Thus, in this study, we used our flat, trilayered, oriented nanofibrous scaffolds to develop trilayered tissue structures with native leaflet orientations through in vivo tissue engineering in a rat model. After 2 months of in vivo tissue engineering, infiltrated cells and their deposited collagen fibrils were found aligned in the circumferential and radial layers, and randomly oriented in the random layer of the scaffolds, i.e., trilayered tissue constructs (TTCs) were developed. Tensile properties of the TTCs were higher than that of the control tissue constructs (without any scaffolds) due to influence of fibers of the scaffolds in tissue engineering. Different extracellular matrix proteins-collagen, glycosaminoglycans, and elastin-that exist in native leaflets were observed in the TTCs. Gene expression of the TTCs indicated that the tissue constructs were in growing stage. There was no sign of calcification in the tissue constructs. The TTCs developed with the flat TN scaffolds indicate that an autologous leaflet-shaped, trilayered tissue construct that can function as a native leaflet can be developed.

UNASSIGNED: The first choice of vascular access for hemodialysis is an autogenous arteriovenous fistula, because prosthetic arteriovenous grafts have a high probability of failure. In this study, Biotubes, in-body tissue architecture-induced autologous collagenous tubes, were evaluated for their potential use as vascular access grafts. Three animal implantation models were developed using beagle dogs, and the in vivo performance of Biotubes was observed after implantation in the acute phase as a pilot study.
UNASSIGNED: Biotubes (internal diameter ca. 4.0 mm, length ca. 5.0 cm, and wall thickness ca. 0.7 mm) were prepared through subcutaneous embedding of specially designed molds in beagle dogs for 8 weeks. The Biotubes were then implanted between the common carotid artery and the jugular vein of beagles via three methods, including side-to-side (in) -end-to-end (out) as type 1 (n = 4), side-to-side (both) as type 2 (n = 4), and side-to-end (in) -end-to-side (out) as type 3 (n = 1 using a composite Biotube).
UNASSIGNED: Although two cases in type 1 and 2 resulted in Biotube deformation, all cases were patent for 4 weeks and maintained a continuous turbulent flow. At 4 weeks after implantation, percutaneous puncture could be performed repeatedly without aneurysm formation or hemorrhage.
UNASSIGNED: Within a short implantation period, with limited animal numbers, this proof-of-concept study showed that Biotubes may have a high potential for use in vascular access.

This study presents the initial 3-year results of the first in-human study of internal shunt restoration using completely autologous vascular grafts, \"Biotubes,\" based on in-body tissue architecture. Biotubes (diameter, 6 mm; length, 7 cm) were prepared as autologous collagenous tubular tissues with approximately 0.5 mm wall thickness by embedding molds (two per patient), assembled with a silicone rod and a stainless steel pipe with many slits, into the patients\' abdominal subcutaneous tissue for 2 months. Two female patients with end-stage renal disease were undergoing hemodialysis with a high probability of failure due to repeated stenosis every few months at the venous outflow regions over 1.5 years. Biotubes formed in both patients and were bypassed over the venous stenosis region of the arteriovenous shunt. After bypass with Biotubes without living cells, palpable thrill and typical turbulent flow pattern were observed by pulsed-wave Doppler. Follow-up angiography showed no signs of dilation or stenosis after implantation, and puncture could be performed easily without graft damage. In both cases, stenosis of Biotubes occurred after 3-4 months. In the first case, percutaneous transluminal angioplasty was not required for over 2 years after implantation even after the development of Biotube stenosis. In the second case, stenosis at the proximal anastomotic site of the Biotube became prominent, and percutaneous transluminal angioplasty was needed 7 months after implantation and then repeated at up to 2 years. This was the first human study successfully supporting the concept of internal shunt restoration for hemodialysis using an autologous Biotube.

In earlier studies, we developed in vivo tissue-engineered, autologous, small-caliber vascular grafts, called \"biotubes,\" which withstand systemic blood pressure and exhibit excellent performance as small-caliber vascular prostheses in animal models. However, biotube preparation takes 4 weeks; therefore, biotubes cannot be applied in emergency situations. Moreover, for responses to various types of surgery, grafts should ideally be readily available in advance. The aim of this study was to develop novel, off-the-shelf, small-caliber vascular grafts by decellularizing in vivo tissue-engineered xenogeneic tubular materials. Silicone rod molds (diameter: 2 mm, length: 70 mm) placed in subcutaneous pouches of a beagle dog for 4 weeks were harvested with their surrounding connective tissues. Tubular connective tissues were obtained after pulling out the impregnated molds. Subsequently, they were decellularized by perfusion with sodium dodecyl sulfate and Triton X-100. They were stored as off-the-shelf grafts at -20°C for 1 week. The decellularized grafts derived from the beagle dog were xenogeneically transplanted to the abdominal aortas of rats (n = 3). No signs of abnormal inflammation or immunological problems due to the xenogeneic material were observed. Echocardiography confirmed the patency of the grafts at 1 month after implantation. Histological evaluation revealed that the grafts formed neointima on the luminal surface, and that the graft walls had cell infiltration. Little accumulation of CD68-positive macrophages in the graft wall was observed. Xenogeneic decellularized tubular tissues functioned as small-caliber vascular grafts, as well as autologous biotubes. This technology enables the easy fabrication of grafts from xenogeneic animals in advance and their storage for at least a week, satisfying the conditions for off-the-shelf grafts.

Electrospinning uses an electric field to produce fine fibers of nano and micron scale diameters from polymer solutions. Despite innovation in jet initiation, jet path control and fiber collection, it is common to only fabricate planar and tubular-shaped electrospun products. For applications that encapsulate cells and tissues inside a porous container, it is useful to develop biocompatible hollow core-containing devices. To this end, by introducing a 3D-printed framework containing a sodium chloride pellet (sacrificial core) as the collector and through post-electrospinning dissolution of the sacrificial core, we demonstrate that hollow core containing polyamide 66 (nylon 66) devices can be easily fabricated for use as cell encapsulation systems. ATR-FTIR and TG/DTA studies were used to verify that the bulk properties of the electrospun device were not altered by contact with the salt pellet during fiber collection. Protein diffusion investigations demonstrated that the capsule allowed free diffusion of model biomolecules (insulin, albumin and Ig G). Cell encapsulation studies with model cell types (fibroblasts and lymphocytes) revealed that the capsule supports the viability of encapsulated cells inside the capsule whilst compartmentalizing immune cells outside of the capsule. Taken together, the use of a salt pellet as a sacrificial core within a 3D printed framework to support fiber collection, as well as the ability to easily remove this core using aqueous dissolution, results in a biocompatible device that can be tailored for use in cell and tissue encapsulation applications.