Effects of viscoelasticity on shear-thickening in dilute suspensions in a viscoelastic fluid.

We investigate previously unclarified effects of fluid elasticity on shear-thickening in dilute suspensions in an Oldroyd-B viscoelastic fluid using a novel direct numerical simulation based on the smoothed profile method. Fluid elasticity is determined by the Weissenberg number Wi and by viscosity ratio 1 - ß = ηp/(ηs + ηp) which measures the coupling between the polymer stress and flow: ηp and ηs are the polymer and solvent viscosity, respectively. As 1 - ß increases, while the stresslet does not change significantly compared to that in the ß â†’ 1 limit, the growth rate of the normalized polymer stress with Wi was suppressed. Analysis of flow and conformation dynamics around a particle for different ß reveals that at large 1 - ß, polymer stress modulates flow, leading to suppression of polymer stretch. This effect of ß on polymer stress development indicates complex coupling between fluid elasticity and flow, and is essential to understand the rheology and hydrodynamic interactions in suspensions in viscoelastic media.

Reductive removal of selenate (VI) in aqueous solution using rhodium metal particles supported on TiO2.

Selenium and its compounds in high concentration are toxic for humans, especially selenate (VI) is the most toxic due to its high solubility in water. To promote the reductive reaction of Se(vi) to Se(iv) or Se(0), which is relatively easy to remove in water, noble metal particles were added as reaction sites with a reductant. The highest removal performance of selenate in aqueous solution was achieved using rhodium particles supported on TiO2 (Rh/TiO2). Selenate was rapidly reduced with hydrazine on the metal particle, leading to a selenium deposition on the particle inhibiting the stable reductive reaction. On the other hand, when a weaker reductant such as formaldehyde was used for the selenate reduction, the selenium deposition was suppressed due to its low reactivity, resulting in a stable reductive reaction of selenate in water.

A bioartificial liver device based on three-dimensional culture of genetically engineered hepatoma cells using hollow fibers.

The bioartificial liver (BAL) device is an extracorporeal liver support system incorporating living hepatocytes. A major problem in BAL device development is to obtain a high number of functional cells. In this study, we focused on a genetically engineered mouse hepatoma cell line, Hepa/8F5, in which elevated liver functions are induced via overexpression of liver-enriched transcription factors activated by doxycycline (Dox) addition. We applied a three-dimensional culture technique using hollow fibers (HFs) to Hepa/8F5 cells. Hepa/8F5 cells responded to Dox addition by reducing their proliferative activity and performing liver-specific functions of ammonia removal and albumin secretion. The functional activities of cells depended on the timing of Dox addition. We also found that Hepa/8F5 cells in the HF culture were highly functional in a low rather than high cell density environment. We further fabricated an HF-type bioreactor with immobilized Hepa/8F5 cells as a BAL device. Although ammonia removal activity of this BAL device was lower than that of the small-scale HF bundle, albumin secretion activity was slightly higher. These results indicated that the BAL device with immobilized Hepa/8F5 cells was highly functional with potential to show curative effects in liver failure treatment.

Evaluation of instability after transtrochanteric anterior rotational osteotomy for nontraumatic osteonecrosis of the femoral head.

BACKGROUND: Transtrochanteric anterior rotational osteotomy results in improvement of joint congruity and prevention of progressive collapse and osteoarthritic changes in patients with femoral head osteonecrosis. However, this procedure remains controversial for patients with extensive collapse due to potential osteoarthritis caused by postoperative instability. The purpose of this study was to evaluate hip instability after osteotomy and determine the relation between instability and radiological and clinical outcomes. METHODS: In all, 27 hips of 24 patients that were followed up for a mean period of 3.8 years were included. Instability was defined as more than 1 mm translation of the femoral head in transverse computed tomography scans obtained at 0 degrees and 45 degrees flexion of the hip joint. Hips were divided into instability and stability groups. RESULTS: Eleven hips (40%) developed instability after surgery. Osteophytes on the femoral head in 10 hips of the instability group and 2 hips of the stability group had increased in size at follow-up. There was a significant relation between postoperative instability and osteophyte formation. Joint space narrowing was not seen in any of the cases. There was no significant difference between the groups in either the postoperative intact ratio of the femoral head or the Japanese Orthopaedic Association hip score. CONCLUSIONS: Neither instability nor osteophyte formation on the femoral head after transtrochanteric anterior rotational osteotomy correlated with progressive osteoarthritic changes or clinical outcome in the presence of an adequate femoral head intact ratio facing the weight-bearing area.

Expansion and differentiation of human iPS cells in a three-dimensional culture using hollow fibers and separation of the specific population by magnetic-activated cell sorting.

In order to employ pluripotent stem cells in the field of regenerative medicine, it is necessary to establish a large-scale culture system for cell differentiation. We have developed a novel three-dimensional method for culturing human induced pluripotent stem (iPS) cells, using hollow fibers (HFs). The cells immobilized inside HFs can proliferate and form multicellular aggregates, capable of achieving a high cell density and promoting further spontaneous cell differentiation. We first cultured human iPS cells for 7 days under conditions that maintained their undifferentiated state and then switched the culture conditions to allow spontaneous cell differentiation. In the 7-day undifferentiated culture, a high cell density of approximately 10-fold that of the initial seeding density was achieved. The upregulation of gene markers for differentiation such as CXCR4 or SOX17 was observed in the culture of differentiated cells. Expression of the lineage-specific cell-surface marker CXCR4 was about 30% at day 5 in the differentiation culture, which was 2-fold higher than that in the traditional monolayer culture. After HF culture, we obtained the CXCR4-positive cell population and performed monolayer culture for further differentiation of the hepatic lineage. In the CXCR4-positive cell population, the expression levels of a few liver-specific gene markers tended to increase. However, there were no significant differences between the separation and non-separation groups, which indicates the need for refinement of the cell separation process and cell maturation procedure in future studies. In conclusion, the HF culture method has potential for achieving the large-scale culturing and spontaneous differentiation of human iPS cells.

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam.

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

[Transtrochanteric rotational osteotomy for the treatment of non-traumatic osteonecrosis of the femoral head].

Non-traumatic osteonecrosis involving the femoral head frequently occurs in young patients especially due to steroids administration or other some reasons. In cases of extensive lesion of the weight-bearing area, collapse is usually progressive. Preservation of the joint in young patients to avoid joint replacement procedures is important and widely accepted. Transtrochanteric rotational osteotomy for the femoral head osteonecrosis is ideal procedure moving the remained viable femoral head area to the loaded portion below the acetabular roof, thus hip joints can be preserved for long term by remodeling even if the stage is progressed preoperatively. This procedure is technically difficult, but should be common in orthopaedic surgeons.

Fabrication of a fiber-type hepatic tissue by bottom-up method using multilayer spheroids.

Liver regenerative medicine, a therapy using cultured hepatocytes or hepatic tissues, has the potential to replace liver transplantation. However, this therapeutic strategy has challenges to overcome, including in construction of the hepatic tissues. As an approach to fabricating functional 3D hepatic tissues, we focused on hepatocyte spheroids, which have high cell density and maintain high liver-specific functions. We employed a bottom-up method using spheroids, arranging hepatocytes and endothelial cells regularly at the time of tissue construction. This enabled a vascular network to be formed within the three-dimensional hepatic tissue. We included NIH/3T3 cells, known to promote vasculature formation by endothelial cells. We fabricated hepatocyte spheroids covered with human umbilical vein endothelial cells (HUVECs) and NIH/3T3 cells (EC-3T3-covered hepatocyte spheroids) and constructed the hepatic tissues by stacking these cell types in hollow fibers. We then performed histological and functional analyses of the resulting hepatic tissues. The hepatic tissues constructed by stacking EC-3T3-covered hepatocyte spheroids showed high liver-specific functions; that is, ammonia removal and albumin secretion. The HUVECs formed endothelial networks. In addition, hypoxia-inducible factor-1α (HIF-1α) expression was suppressed in the hepatic tissue throughout the culture period and the hepatic tissue was sufficiently strong for use in certain analyses and applications. In summary, we fabricated a functional 3D hepatic tissue by the bottom-up method using hepatocyte spheroids covered with HUVECs and NIH/3T3 cells.

Posterior rotational osteotomy for nontraumatic osteonecrosis with extensive collapsed lesions in young patients.

BACKGROUND: In young patients with nontraumatic femoral head osteonecrosis with extensive and collapsed lesions, joint preservation is a goal if total joint arthroplasty is to be avoided. We evaluated the effectiveness of a posterior rotational osteotomy in this patient population. METHODS: We reviewed thirty-five hips in twenty-eight young patients with nontraumatic femoral head osteonecrosis treated by posterior femoral neck rotational osteotomy. All femoral heads were collapsed, and seven hips showed joint-space narrowing. Lateral radiographs of the femoral head revealed that 15% of the mean posterior portion and 17% of the mean anterior portion of the femoral head consisted of radiographically apparent living bone. The mean age of the patients (ten women and eighteen men) was twenty-eight years. The mean follow-up period was eight years. RESULTS: Less than six months after surgery, the radiographically apparent area of living bone of the femoral head below the acetabular roof was shown to be 59% on the standard anteroposterior radiograph and 54% on the 45 degrees -flexion radiograph. In thirty-three hips (94%), further collapse of the femoral head was prevented and an adequate amount of living bone was demonstrated on the loaded lateral portion of the femoral head on the final follow-up radiographs. Progressive joint-space narrowing was seen in four hips. CONCLUSIONS: In young patients with osteonecrosis and extensively collapsed lesions of the femoral head, posterior femoral neck rotational osteotomy appears to be effective in delaying the progression of degeneration if an adequate area of living bone can be placed under the loaded lateral portion of the acetabulum. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions to Authors on jbjs.org for a complete description of levels of evidence.

Formation of three-dimensional hepatic tissue by the bottom-up method using spheroids.

Liver regenerative medicine has attracted attention as a possible alternative to organ transplantation. To address the challenge of liver regenerative medicine, the development of a construction method has been proposed for liver tissue in vitro with a high cell density and high functionality for transplantation into patients with severe liver failure. In this study, we fabricated highly functional three-dimensional hepatic tissue by a bottom-up method using spheroids. The hepatic tissue was formed by stacking hepatocyte spheroids covered with human umbilical vein endothelial cells (HUVECs). Hepatic tissue constructs were evaluated for cell survival, liver-specific functions, and histologically. As a result, we identified improvements in liver-specific functions (ammonia removal and albumin secretion) and cell survival. In addition, HUVECs were regularly distributed at every 100 µm within the tissue, and live cells were present within the whole tissue construct throughout the culture period. In summary, we successfully fabricated highly functional hepatic tissue by the bottom-up method using HUVEC-covered hepatocyte spheroids.

Early repair of necrotic lesion of the femoral head after high-degree posterior rotational osteotomy in young patients-a study evaluated by volume measurement using magnetic resonance imaging.

We investigated the repair of femoral head necrosis with extensive necrotic lesions treated by high-degree posterior rotational osteotomy (HDPRO) in young adults and adolescents (mean age; 30.8 years) using magnetic resonance imaging (MRI). HDPRO was performed on 72 hips from 66 cases, and of those, 60 hips from 60 cases were included in this study for data analysis. All cases had extensive collapsed lesion preoperative anteroposterior radiographs. In total, 34 hips were male and 26 were females. In total, 19 had a history of steroid administration, 11 had a previous femoral neck fracture, 7 had no particular etiologic factor, and 4 had a followed slipped capital femoral epiphysis. Antero-inferior viable areas were transferred to the loaded portion below the acetabular roof by this operation. The mean posterior rotational angle was 118.5°. MRI was taken after 1 month, 6 months and 1-year post-operatively. Post-operative necrotic lesion volume compared with the preoperative necrotic lesion volume was defined as lesion volume ratio (%). The reduction of necrotic volume was observed over time, and at 1 year post-operatively, it was 19.4% for patients in their teens, 35.3% for those in twenties, 42.8% for those in their thirties and 59.5% for those in their forties. From this study, we concluded that the extensive necrotic lesions decreased in size within a short period after HDPRO in young patients.

Evaluation of a hybrid artificial liver module based on a spheroid culture system of embryonic stem cell-derived hepatic cells.

Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

Hepatic differentiation of mouse embryonic stem cells and induced pluripotent stem cells during organoid formation in hollow fibers.

We have focused on pluripotent stem cells as a potential source of a hybrid-type artificial liver (HAL) and tried to develop a method for differentiating the pluripotent stem cells into cells of a hepatic lineage. In this study, we investigated the hepatic differentiation of mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells by applying hollow fiber (HF)/organoid culture method, in which cultured cells form a cellular aggregate called an "organoid" in the lumen of the HF. ES and iPS cells were injected into HFs to induce organoid formation, and cells were cultured. To induce hepatic differentiation, we added differentiation-promoting agents to the culture medium. The expression levels of differentiation-related genes were up-regulated, with cell proliferation and organoid formation inside HFs. Since we were able to achieve a high cell density in culture, the maximum levels of liver-specific functions per unit volume in the differentiating ES and iPS cells reached a level comparable to or better than that of primary mouse hepatocytes. In conclusion, ES and iPS cells have the potential to be a cell source for a HAL, and the HF/organoid culture method, therefore, has promise as a basic technology for the development of a HAL.

In vitro reconstruction of a three-dimensional mouse hematopoietic microenvironment in the pore of polyurethane foam.

Hematopoietic stem cells exist in specific niches in the bone marrow, and generate either more stem cells or differentiated hematopoietic progeny. In such microenvironments, cell-cell and cell-matrix interactions are as important as soluble factors such as cytokines. To provide a similar environment for in vitro studies, a three-dimensional culture technique is necessary. In this manuscript, we report the development of a three-dimensional culture system for murine bone marrow mononuclear cells (mBMMNCs) using polyurethane foam (PUF) as a scaffold. The mBMMNCs were inoculated into two kinds of PUF disks with different surface properties, and cultured without exogenous growth factors. After seeding the inside of the PUF pores with mBMMNCs, PUF disks were capable of supporting adherent cell growth and continuous cell production for up to 90 days. On days 21-24, most nonadherent cells were CD45 positive, and some of the cells were of the erythroid type. From comparisons of the cell growth in each PUF material, the mBMMNC culture in PUF-W1 produced more cells than the PUF-R4 culture. However, the mBMMNC culture in PUF-W1 had no advantages over PUF-R4 with regard to the maintenance of immature hematopoietic cells. The results of scanning electron microscopy and colony-forming assays confirmed the value of the different three dimensional cultures.

An approach for formation of vascularized liver tissue by endothelial cell-covered hepatocyte spheroid integration.

Tissue vascularization in vitro is necessary for cell transplantation and is a major challenge in tissue engineering. To construct large and regularly vascularized tissue, we focused on the integration of endothelial cell-covered spheroids. Primary rat hepatocytes were cultured on a rotary shaker, and 100-150 mum spheroids were obtained by filtration. The hepatocyte spheroids were coated with collagen by conjugation with a type 1 collagen solution. Collagen-coated hepatocyte spheroids were cocultured with human umbilical vein endothelial cells (HUVECs), and monolayered HUVEC-covered hepatocyte spheroids were constructed. Without a collagen coat, many HUVECs invaded hepatocyte spheroids but did not cover the spheroid surface. To construct regularly vascularized tissue, we packed HUVEC-covered hepatocyte spheroids in hollow fibers used for plasma separation. Packed spheroids attached to each other forming a large cellular tissue with regular distribution of HUVECs. At day 9 after packing, HUVECs invaded the hepatocyte spheroids and a dense vascular network was constructed. Collagen coating of spheroids is useful for the formation of endothelial cell-covered spheroids and subsequent regular vascularized tissue construction.

Respherical contour with medial collapsed femoral head necrosis after high-degree posterior rotational osteotomy in young patients with extensive necrosis.

Osteonecrosis of the femoral head often occurs in patients under the age of 50 years. In this study, the authors evaluated the effectiveness of high-degree posterior rotation in terms of regaining the spherical contour of severely collapsed necrotic femoral head that was moved medially. They also investigated whether or not subchondral fracture disappeared on the medial femoral head on postoperative anteroposterior radiographs as a result of remodeling after this procedure.

Evaluation of a bioreactor with stacked sheet shaped organoids of primary hepatocytes.

Hepatocyte organoids have an in vivo-like cell morphology and maintain cell viability and function in vitro. On the other hand, the oxygen supply to hepatocytes is sometimes limited in the core of organoids that are more than 100 mum in thickness. In this study, we designed and examined a new bioreactor using sheet-shaped organoids (organoid-sheets) in which the thickness was controlled to prevent hepatocyte death in the core of organoid due to limitation of oxygen supply. The cell culture space consisted of stacked organoid formation spaces and medium flow channels. Each space was separated by flat porous polycarbonate membranes, and the organoid thickness was controlled at 100 microm with a stainless steel spacer. Freshly isolated hepatocytes (7.0 x 10(7)) were immobilized in the bioreactor, yielding a cell density of 4.5 x 10(7) cells/cm(3)-bioreactor. Of the five flow rates tested (1.0, 5.0, 10, 20, and 50 mL/min), the bioreactor with the 10 mL/min had the highest ammonia removal and albumin secretion activities for at least 14 days. In conclusion, a new bioreactor controlling organoid thickness is useful for achieving high cell density culture and the maintenance of hepatocyte function to avoid cell death in the core of the organoids due to limitation of oxygen supply. The bioreactor may be useful for the development of various applications using cultured hepatocytes.

Reconstruction of cartilage tissue using scaffold-free organoid culture technique.

We applied the scaffold-free culture method to chondrocytes and attempted to reconstitute articular cartilage grafts. Primary rat costal chondrocytes were immobilized into hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm(3) to induce the formation of cylindrical-shaped multicellular aggregates (organoids) and cultured for one month. The organoids were evaluated by histological and gene expression analyses. Chondrocytes formed cylindrical organoids in hollow fibers (HFs). Histochemical analysis revealed the accumulation of a cartilage extracellular matrix (collagen and proteoglycan) around cells in the lumen of HFs with culture time, forming a low-cellular-density tissue similar to native cartilage by day 28. Furthermore, in contrast to that in traditional monolayer culture, the organoid maintained the gene expression of the cartilage extracellular matrix (type II collagen, aggrecan) for one month of culture. In conclusion, our organoid formation method was effective in producing a cartilage-like tissue. This result suggests that the technique may be applicable to the development of an articular cartilage graft.

A new culture technique for hepatocyte organoid formation and long-term maintenance of liver-specific functions.

To develop a useful hybrid artificial liver, it is important to use cultured hepatocytes that maintain liver-specific functions for a long time. These requirements were achieved recently by the use of a hepatocyte multicellular aggregate (organoid) with a tissue-like structure. In this study, we developed a three-dimensional culture of hepatocytes that formed an organoid. Primary rat hepatocytes were immobilized inside hollow fibers (for plasma separation) by centrifugation. Hepatocytes formed a cylindrical organoid (cylindroid) of 200 mum in diameter by day 2 of culture. We used two types of culture media, medium A (Williams' medium E containing insulin and epidermal growth factor) and medium B (Dulbecco's modified Eagle's medium containing insulin, epidermal growth factor, and hydrocortisone). In medium A, the hepatocyte cylindroid diminished after 14 days of culture and liver-specific functions of the hepatocyte cylindroid nearly disappeared after 1 month of culture. In contrast, hepatocyte cylindroid cultured in medium B maintained its morphology and liver-specific functions for 2-5 months. These results indicate that a combination of the new culture technique and suitable culture medium is effective for expression and maintenance of liver-specific functions of hepatocytes. This culture technique will be helpful in the development of a hybrid artificial liver.