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Background Late-life depressive disorder is becoming an important issue in health economics in the world, as it has been reported to be one of major risk factors for incidence of dementia. Identification of predictive markers associated with depression in later life is therefore of high priority in public health. The aim of the study was to examine the association of salivary cortisol levels with a later depressive state in elderly healthy people living in a rural Japan community. Methods Salivary cortisol levels were measured in 68 elderly healthy people (24 men; 44 women) followed by completion of the BDI, MMSE, and FAB from 2004 to 2006. The same cohort underwent BDI again from 2007 to 2009. Results In healthy elderly women subject, a significant positive correlation was found between salivary cortisol levels at baseline and BDI scores at follow-up, but not at baseline. Salivary cortisol levels at baseline were not correlated with the score of either MMSE or FAB. When the cut-off point of BDI scores were set at 20/21, logistic regression analyses revealed that salivary cortisol levels at baseline had a significant positive relationship with a later depressive state. Age and gender were also significantly related with a later depressive state. Limitations The present study involves small number of participants. Conclusions Higher salivary cortisol levels were associated with a later depressive state in elderly healthy women living in rural community. Salivary cortisol might be a predictive marker for a later depressive state in elderly women.

Porous scaffolds play important roles in tissue engineering. Biodegradable synthetic polymers, such as poly( l -lactic acid) (PLLA), frequently are used in the preparation of porous scaffolds. Pretreating the surface of a PLLA porous scaffold is required to increase its wettability for smooth cell seeding due to the hydrophobic property of the scaffold's surface. In this study, a simple coating method was used to modify the surface of the PLLA sponges. The coating method included three steps: filling the PLLA sponge pores with collagen aqueous solution, centrifuging to remove excess collagen, and, finally, freeze-drying. Compared with the uncoated PLLA sponge, the collagen-coated PLLA sponge demonstrated both improved wettability and high water absorption. Cells were smoothly seeded in the collagen-coated PLLA sponges by dropping a cell suspension solution onto the sponges. Cells adhered to the collagen-coated sponge and were distributed homogeneously throughout the collagen-coated PLLA sponge.

A two-dimensional microarray of ten thousand (100×100) hepatocyte hetero-spheroids, underlaid with non-parenchymal cells, was successfully constructed with a 100-μm spacing on micro-fabricated glass substrates that were coated with poly(ethylene glycol) (PEG). Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100-μm, functioning as a miniaturized liver to secrete albumin for at least 1 month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors (TBB and CBB), detecting a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.

The structural changes and degradation behaviors of poly(L-lactic acid) (PLLA) and poly(e-caprolactone) (PCL) sponges were studied by incubation in phosphate buffer solution and compared with those of previously reported poly(lactic-co-glycolic acid) (PLGA) sponge. The changes of pH, weight, molecular weight, crystallinity, and thermal properties of glass transition and melting were measured. The influence of incubation temperature on the structural change of each type of sponge was investigated in details. During incubation, the PLLA sponge showed secondary crystallization, the PCL sponge showed lamellar thickening, and the PLGA sponge showed physical aging. Depending on the type of biodegradable polymers, the incubation temperature caused different structural changes during the incubation and the structural changes influenced several degradation behaviors. POLYM. ENG. SCI., 50:1895–1903, 2010. © 2010 Society of Plastics Engineers

The development of porous scaffolds having both high porosity and strong mechanical strength for tissue engineering and regenerative medicine has been quite challenging. A novel hybrid poly(L-lactic acid) (PLLA)-collagen hybrid sponge was developed by enclosing collagen sponge in a cup-shaped PLLA sponge to meet the necessary requirements. Collagen sponge was formed in the center of the PLLA sponge cup, and collagen microsponges were formed in the pores of the PLLA sponge cup. The PLLA-collagen hybrid sponge showed higher mechanical strength than did those of the PLLA sponge cup and collagen sponge. The porosity of the PLLA-collagen hybrid sponge was greater than that of the PLLA sponge cup. The cup-shaped PLLA sponge skeleton provided the hybrid sponge with high mechanical strength and protected against cell leakage during cell seeding, while the central collagen sponge contributed to high porosity, and facilitated cell adhesion and distribution in the hybrid sponge. Cartilaginous tissue was successfully regenerated when chondrocytes were cultured in the hybrid sponge. This method of hybridization will provide a new technique for the preparation of functional porous scaffolds for tissue engineering.

A new type of collagen sponge was prepared as a tissue engineering scaffold using ice particulates as a template. The sponge has a hierarchical structure of large open pores on the top surface and interconnected small pores in the inner bulk body. The shape, size, and density of the surface large pores were determined by the ice particulates that were used as the template while the interconnected small pores were determined by the freezing temperature. The open and interconnected porous structure of the new collagen sponge facilitated cell seeding, cell penetration, and distribution throughout the scaffold, and accelerated cell proliferation and regeneration of new tissue. These ice particulate templates could be used to create open and interconnected porous scaffold structures.

癌や老化などのさまざまな疾患では，過剰な活性酸素(O2-・)の発生が深く関係している．生体内で過剰に発生した O2-・ を消去するため，生体内投与を視野に入れ，かつ，長期血中滞留性を有する新規抗酸化剤としてカチオン性マンガンポルフィリン錯体導入ニオソームを調製した．抗酸化作用を有するマンガン 5,10,15,20-テトラキス(N-2-メチルピリジル)ポルフィリン(MnT2MePyP)およびマンガン 5,10,15,20-テトラキス(S,S-4-ジメチルスルフォニオフェニル)ポルフィリン(MnT4Me2SuP)をノニオン性界面活性剤主成分(プルロニック(Pluronic)F-88/コレステロール系)のニオソームに導入した(マンガンポルフィリン/ニオソーム)．調製したマンガンポルフィリン/ニオソームの構造は，動的光散乱および蛍光偏光解消法により確認した．血中投与した際の安定性は棚置き安定性試験および擬似血中滞留試験により評価した．また，抗酸化剤としての性能をストップトフロー法および動物試験により評価した．ストップトフロー法の結果より，マンガンポルフィリン/ニオソームはマンガンポルフィリン単独系とほぼ同等の高い抗酸化活性を示した．マウスを用いた動物実験の結果より，マンガンポルフィリン/ニオソームはマンガンポルフィリン錯体単独系よりも長期血中滞留性を有し，有意な抗酸化活性の向上が見られた．マンガンポルフィリン/ニオソームは抗酸化作用の DDS への応用が期待できる．

As excellent scaffolds for cultivating bone cells, porous beads of bioactive ceramics such as HAp, TCP are considered to be promising. HAp and α-TCP are well known as non-toxic bioceramics to human cells, but their behavior in living body fluid are different. HAp is bioactive material which has both high strength and better tissue-adhesive properties, but that is not readily absorbed by the human body. On the contrary, α-TCP is highly bioabsorbable; it is quickly absorbed by the body, and, therefore, disappears before bone is completely replaced. To realize a composite which has suitable solubility in living body fluid, α-TCP/ HAp functionally graded porous beads were fabricated by the method of spheroidization in liquid nitrogen. This type of composite maintains the function of scaffold with sufficient strength up to growth of new bone, and after the growth, it is expected to absorbed completely in the body. In the present study, ceramic beads with α-TCP at the center were fabricated and coated with a functionally graded layer of HAp. By controlling the thickness of HAp layer, which could be realized by changing time of hydrolytic reaction, the absorption rate into the body would be easily controlled. In addition, to accelerate the formation of porous structure, some acid solutions were used to dissolve the beads surface and to penetrate pores toward inside of the beads. Observed surface and inner structure by SEM, also the measured change in absorption rate will be presented.

Atomic force microscopy (AFM) is a powerful tool in imaging cells and tissues and probing their mechanical properties. Articular chondrocytes, the cells responsible for the production and maintenance of cartilaginous extracellular matrix in the knee joint, change their morphology and dedifferentiate during in vitro expansion culture. It was unclear if the mechanical properties of chondrocytes change accompanying phenotype variation. The elasticity of in vitro serially cultured bovine articular chondrocytes was investigated using AFM. The chondrocytes changed their morphology from round to spindle-like. The freeze-dried P0 chondrocytes showed significantly higher modulus than did the serially passaged (P1–P4) chondrocytes. The change of chondrocyte morphology was accompanied with a decrease of elastic modulus.

Microenvironments, composed of many kinds of cytokines and growth factors plus extracellular matrices with diverse electrostatic properties, play key roles in controlling cell functions in vivo. In this study, three kinds of water-soluble polymers, positively charged poly(L-lysine) (PLL), negatively charged poly(acrylic acid) (PAAc) and neutral poly(ethylene glycol) (PEG), were compared based on their effects on the adhesion, spread, proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). The MSCs were seeded and cultured in the presence of polymers of different concentrations applied by methods using coating, mixing or covering. The effects of the water-soluble polymers depended on their electrostatic properties and method of application. The methods were in the order of coating, mixing and covering in terms of high to low influence. A low concentration of PLL promoted MSC adhesion, spread, proliferation and chondrogenic differentiation, while a high concentration of PLL was toxic. The PEG-coated surface facilitated cell aggregation and spheroid formation by inhibiting cell adhesion. A high concentration of mixed PEG (10 microg/ml) promoted cell proliferation in serum-free medium. PAAc showed no obvious effects on MSC adhesion, spread, proliferation, or chondrogenic differentiation.

Three-dimensional cultures of pancreatic islet cells in porous scaffolds or hydrogels have been constructed as a biohybrid artificial pancreas. A thin mesh of a PLGA-collagen hybrid was used to culture rat RIN-5F cells. The hybrid mesh was coated with laminin, fibronectin, vitronectin, type IV collagen, and poly(L-lysine) were evaluated and mesh without coating was used as a control. Cell adhered and proliferated on all of the coated and uncoated meshes. The cells formed spheroids in the uncoated, poly(L-lysine)-, fibronectin-, vitronectin-, and type IV collagen-coated hybrid meshes, while forming a layered structure in the laminin-coated hybrid mesh. Cell adhesion on the coated PLGA-collagen hybrid meshes was higher than that for the uncoated hybrid mesh. The laminin-coated hybrid mesh showed the greatest level of adhesion. The insulin secretion capacity of RIN-5F cells was at the same level for all coated and uncoated PLGA-collagen hybrid meshes and higher than that of cells cultured on cell culture plates. The 3D cultured PLGA-collagen hybrid meshes promoted insulin production capacity. Gene expression analysis showed that genes encoding insulin I, insulin II, and the pancreatic transcription factor PDX-1 (pancreas/duodenum homeobox 1) was expressed. These results indicate that the PLGA-collagen hybrid meshes support adhesion, proliferation, and differentiation of RIN-5F cells that allows culturing pancreatic islet cells on 3D constructs.

Polystyrene cell-culture plates modified with positively charged polyallylamine (PAAm) and negatively charged poly(acrylic acid) (PAAc) and unmodified plate were used for the culture of human mesenchymal stem cells (MSCs) to study the effect of surface electrostatic properties on their osteogenic differentiation. All of these surfaces supported cell adhesion and proliferation. However, the cells adhered, spread, and proliferated somewhat more quickly on the PAAm-modified surface than they did on the PAAc-modified and control surfaces. Osteogenic differentiation was examined by alkaline phosphatase (ALP) staining, alizarin red S staining, and gene-expression analysis. The MSCs cultured on the three kinds of surfaces in the presence of dexamethasone were positively stained with ALP and alizarin red S staining, while the cells cultured without dexamethasone were not positively stained. Gene-expression analyses using real-time PCR indicated that MSCs cultured on these surfaces in the presence of dexamethasone expressed osteogenic marker genes, encoding ALP, osteocalcin, bone sialoprotein, osteopontin, and type I collagen. These results indicate that the positively charged, negatively charged, and unmodified surfaces supported osteogenic differentiation, and that their effect required the synergistic effect of dexamethasone.

Objective: For regeneration of bone in a clinical setting, an adequate number of committed osteoblasts is required. Bone marrow mesenchymal stromal cells could provide a source of osteoblasts for bone regeneration. In addition, it is well known that mesenchymal cell condensation plays an important role in the early stages of membranous osteogenesis, and dynamic flow can positively influence and enhance osteodifferentiation of mesenchymal stromal cells. Therefore, to imitate bone development, we adopted a rotation culture method rather than a static culture method. Materials and Methods: Osteogenic differentiation was evaluated by histological, immunochemical and reverse transcriptase-polymerase chain reaction analyses. Results: Cell condensation and a 3-dimensional dynamic environment in a rotation culture may enhance osteoblastic phenotype development and mineralised matrix synthesis in vitro. The extracellular matrix of cell aggregates was endogenously synthesised in an entirely natural manner by the cells themselves. The cell aggregates by rotation culture showed mineral deposition and osteocalcin expression at 7 days, whereas these effects were not detected in the static culture until 14 days. Conclusion: These findings indicate that, compared with static culture in well plates, rotation culture provides a more suitable culture environment that is much closer to the actual cellular environment in vivo.

A quantitative flow visualization study of a scaled-up model of a centrifugal blood pump was performed. Since the size of the scaled-up model was three times as large as the original pump under development, and the kinematic viscosity of the saline solution used as the working fluid was approximately one-third that of the blood, we obtained a similar flow at one twenty-seventh the angular velocity of the original pump. The flow was visualized by seeding the saline solution with neutrally buoyant particles and by illuminating the model with a laser light sheet. Since the gap flow behind the impeller is important for thrombus formation, it was recorded by a high-speed video camera, and the velocity field was evaluated automatically by particle tracking velocimetry. It was shown that in the gap behind the impeller there existed a region where the velocity profile was almost flat which can be called a core region. The results indicated the effectiveness of the present visualization technique for centrifugal blood pumps.

Cartilaginous gene expression decreased when chondrocytes were expanded on cell-culture plates. Understanding the dedifferentiation mechanism may provide valuable insight into cartilage tissue engineering. Here, we demonstrated the relationship between the nuclear shape and gene expression during in vitro expansion culture of chondrocytes. Specifically, the projected nuclear area increased and cartilaginous gene expressions decreased during in vitro expansion culture. When the nuclear deformation was recovered by cytochalasin D treatment, aggrecan expression was up-regulated and type I collagen (Col1a2) expression was down-regulated. These results suggest that nuclear deformation may be one of the mechanisms for chondrocyte dedifferentiation during in vitro expansion culture.

Three kinds of polyelectrolyte complex membranes were prepared by mixing chitosan and DNA at the ratios of 3:1, 1:1, and 1:3. The effects of the membranes on cell adhesion, spreading, and viability were investigated. The membrane with the high ratio of chitosan had a less hydrophilic surface. The surface of the polyelectrolyte complex membranes became rough as the DNA content increased. The ratio of DNA to chitosan showed some effect on cell adhesion, spreading, and viability. Human mesenchymal stem cells adhered and spread on membranes prepared at chitosan/DNA ratios of 1:1 and 3:1, while they did not on membranes prepared at a chitosan/DNA ratio of 1:3. Cells aggregated on the membrane prepared at a chitosan/DNA ratio of 1:3. Cell viability was also higher on membranes prepared at chitosan/ DNA ratios of 1:1 and 3:1 than that on the membrane prepared at a chitosan/DNA ratio of 1:3. The membrane with a high content of chitosan facilitated cell adhesion and spreading, while a high content of DNA suppressed cell adhesion and spreading.

Cationic gold nanoparticles modified with 2-aminoethanethiol, 8-amino-1-octanethiol, and 11-amino-1- undecanethiol were prepared by NaBH4 reduction of HAuCl4 in the presence of thiols in water or a water/ethanol mixture solvent. The formation of gold nanoparticles depended on the kind of solvent, and the concentration of NaBH4, HAuCl4, and thiols. A low concentration of HAuCl4 and a high concentration of NaBH4 facilitated the formation of gold nanoparticles. A high concentration of HAuCl4 solution resulted in aggregation and a low concentration of NaBH4 did not induce the formation of gold nanoparticles. The size of the gold nanoparticles increased with an increase of the concentration of the HAuCl4 solution, while it decreased with an increase of the concentration of the NaBH4 solution. When thiols were present, the HAuCl4/thiol ratio must be in the range of 1:1 to 1:2 for the formation of gold nanoparticles in both the water and water/ethanol mixture solvents. Modification with thiol molecules changed surface charge of the gold nanoparticles from negative to positive, and stabilized the gold nanoparticles. The cationic gold nanoparticles promoted gene transfection. A high ratio of gold nanoparticles to DNA increased transfection efficiency. However, the length of the alkyl groups had no evident effect.

The effects of extracellular matrix (ECM) proteins and cationic polymers on the adhesion and proliferation of rat islet cells, RIN-5F cells, were investigated. ECM proteins of laminin, fibronectin, vitronectin, type I collagen, type II collagen, and type IV collagen, and cationic polyelectrolytes of poly(L-lysine) and poly(allylamine) were coated on the wells of polystyrene cell culture plates. Their effects on the adhesion and proliferation of RIN-5F in serum-free and serum mediums were compared. The cell number on the laminin-coated surface was the highest among the coated surfaces. Laminin promoted cell adhesion more strongly than did the other ECM proteins and cationic polyelectrolytes. Vitronectin, type IV collagen, and poly(L-lysine) showed moderate effects, but type I collagen and type II collagen did not have any effects on adhesion and proliferation compared with the uncoated polystyrene cell culture plate. Fibronectin promoted cell adhesion but not cell proliferation. Cationic poly(allylamine) had an inhibitory effect in serum-free medium and for longterm culture in serum medium. The ECM proteins of laminin, vitronectin, and type IV collagen, and cationic poly(Llysine) will be useful for the surface modification and construction of biomaterials and scaffolds for islet cell culture and tissue engineering.

To noninvasively evaluate the connection states of collagen fiber, a characterizing factor of the physical property, is considered to be helpful in the evaluation of cartilage functions. The purpose of this study was to examine how the connection states of collagen influence the MRI parameters by evaluating the collagen gel with various connection states using MRI. MRI was performed to six type I collagen gel samples with various connection status and a water sample. The evaluation parameters included T1 relaxation time, T2 relaxation time, and diffusion coefficient. With regard to gel samples with cross-links, the T2 relaxation time was shortened in proportion to the dose of glutaraldehyde. It is considered that as the glutaraldehyde concentration increases, the distance between protons in water molecules decreases; this is followed by a stronger bipole–bipole interaction, resulting in a shorter T2 relaxation time. The diffusion coefficient for gel samples with cross-links also decreased with increasing glutaraldehyde concentrations. However, gel samples without glutaraldehyde were almost the same as that of the water. This result suggested that the degree of entrapment of water inside the gel samples without cross-links, even when it converted into gel, was found to be nearly equal to that of the free water.

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.

Human mesenchymal stem cells (MSCs) were cultured on polystyrene surfaces modified with photoreactive azidophenyl-derivatives of three different chargeable polymers, poly(acrylic acid) (PAAc), polyallylamine (PAAm), and poly(ethylene glycol) (PEG). The MSCs adhered and spread both on a PAAm-modified surface and on PAAc-modified and polystyrene (control) surfaces. However, the cells adhered more easily to the PAAm-modified surface. The MSCs did not attach to the PEG-modified surface and aggregated to form pellets immediately after cell seeding. The cells proliferated on the PAAc-, PAAm-modified and control surfaces with culture time, formed a monolayer, and aggregated to form pellets. The cells in the pellets that formed on the PAAm- and PEG-modified surfaces after 2 weeks culture had a round morphology and the extracellular matrices were positively stained by safranin O and toluidine blue, while those that formed on the PAAc-modified and control surfaces had a spindle, fibroblast-like morphology and were not positively stained by safranin O and toluidine blue. The pellets that formed on the PAAm- and PEG-modified surfaces contained significantly higher levels of sulfated glycosaminoglycans than did those that formed on the PAAc-modified and control surfaces. Type II collagen and cartilage proteoglycan were immunohistologically detected in the pellets that formed on PAAm- and PEG-modified surfaces, but not those that formed on the PAAc-modified and control surfaces. The MSCs cultured on the PAAm- and PEG-modified surfaces expressed a high level of cartilaginous genes encoding type II collagen and aggrecan, while the MSCs cultured on the PAAc-modified and control surfaces did not express these genes. These results suggest that the PAAm-modified surface supported cell adhesion and proliferation and also promoted chondrogenic differentiation of the MSCs. The PAAc-modified and polystyrene surfaces supported cell adhesion and proliferation, but not chondrogenic differentiation. The PEG-modified surfaces did not support cell adhesion, but did promote chondrogenic differentiation. The adhesion, proliferation, and differentiation of the MSCs could be controlled by surface chemistry.

A three-dimensional culture of mesenchymal stem cells (MSCs) in a porous scaffold has been developed as a promising strategy for cartilage tissue engineering. The chondrogenic differentiation of MSCs derived from human bone marrow was studied by culturing the cells in a novel scaffold constructed of leakproof collagen sponge. All the surfaces of the collagen sponge except the top were wrapped with a membrane that has pores smaller than the cells to protect against cell leakage during cell seeding. The cells adhered to the collagen, distributed evenly, and proliferated to fill the spaces in the sponge. Cell seeding efficiency was greater than 95%. The MSCs cultured in the collagen sponge in the presence of TGF-β3 and BMP6 expressed a high level of genes encoding type II and type X collagen, sox9, and aggrecan. Histological examination by HE staining indicated that the differentiated cells showed a round morphology. The extracellular matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. These results suggest the chondrogenic differentiation of MSCs when cultured in the collagen sponge in the presence of TGF-β3 and BMP6.

Linear and Star-shaped PCL-b-PEG copolymers were synthesized through a two step process, including the first step to synthesize the star-shaped PCL through ring-opening polymerization of Γ-caprolactone initiated from multi-functional alcohol under the existence of tin(II) ethylhexanoate [Sn(Oct)2] catalyst, and the following step to couple the obtained star-shaped PCL with PEG segments using bi-functional linker. The structure of the polymers was confirmed by IR, NMR, GPC, et al. The aggregation behaviors of the star-shape copolymers were compared with that of the linear block copolymer with corresponding molecular weight of each arm, and the influences of structure factors were discussed.

Dehydroepiandrosterone (DHEA), a precursor of sex steroid hormones, is synthesized by cholesterol side-chain cleavage cytochrome P-450 and 17α-hydroxylase cytochrome P-450 mainly from cholesterol and converted to testosterone and estrogen by 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD, and aromatase cytochrome P-450. Although sex steroid hormones have important effects in the protection of articular cartilage, it is unclear whether articular cartilage has a local steroidogenic enzymatic machinery capable of metabolizing DHEA. This study was aimed to clarify whether steroidogenesis-related enzymes are expressed in articular chondrocytes, whether expression levels are changed by DHEA, and whether articular chondrocytes are capable of synthesizing sex steroid hormones from DHEA. Articular chondrocytes isolated from adult rats were cultured with DHEA for 3 days. All of the mRNA expressions of steroidogenesis-related enzymes were detected in cultured articular chondrocytes of rats, but the mRNA expression levels of testosterone and estradiol in cultured media increased after the addition of DHEA. These findings provided the first evidence that articular chondrocytes expressed steroidogenesis-related enzyme genes and that they are capable of locally synthesizing sex steroid hormones locally from DHEA.

Hydrostatic pressure is one of the most frequently used mechanical stimuli in chondrocyte experiments. A variety of hydrostatic pressure loading devices have been used in cartilage cell experiments. However, no gas-controlled system with other than a low pressure load was used up to this time. Hence we used a polyolefin bag from which gas penetration was confirmed. Chondrocytes were extracted from bovine normal knee joint cartilage. After 3 passages, dedifferentiated chondrocytes were applied to form a pellet. These pellets were cultured in chemically defined serum-free medium with ITS+Premix for 3 days. Then 5 MPa of cyclic hydrostatic pressure was applied at 0.5 Hz for 4 h per day for 4 days. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 5-fold increase in the levels of aggrecan mRNA due to cyclic hydrostatic pressure load (p

A novel grid pattern of two kinds of nanothick microgels was developed by alternate patterning using photolithography. At first, 100-microm-wide nanothick PAAm microgel stripes were grafted on a polystyrene surface by UV irradiation of the photoreactive azidobenzoyl-derivatized polyallylamine-coated surface through a photomask with 100-microm-wide stripes. Then, a second set of 100-microm-wide nanothick PAAc microgel stripes were grafted across the PAAm-grated polystyrene surface by UV irradiation of the photoreactive azidophenyl-derivatized poly(acrylic acid)-coated surface through a photomask placed perpendicularly to the first set of PAAm microgel stripes. The PAAc microgel stripe pattern was formed over the PAAm microgel stripe pattern. The cross angle of the two microgel stripes could be controlled by adjusting the position of the photomask when the second microgel pattern was prepared. Swelling and shrinking of the microgels were investigated by scanning probe microscopy (SPM) in an aqueous solution. SPM observation indicated that the thickness of the gel network was 100 to 500 nm. The regions containing PAAm, PAAc, and the PAAc-PAAm overlapping microgels showed different swelling and shrinking properties when the pH was changed. The PAAm microgel swelled at low pH and shrank at high pH whereas the PAAc microgel swelled at high pH and shrank at low pH. However, the PAAc-PAAm overlapping microgel did not change as significantly as did the two microgels, indicating that the swelling and shrinking of the two gels was partially offset. The pH-induced structural change was repeatedly reversible. The novel grid pattern of nanothick microgels will find applications in various fields such as smart actuators, artificial muscles, sensors, and drug delivery systems as well as in tissue engineering and so forth.

A biocompatible glue consisting of human serum albumin (HSA) and citric acid derivative (CAD), named CAD-A glue was developed. CAD was successfully synthesized by the reaction between citric acid and N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride. When the CAD-A glue was applied to the collagenbased casings, it took 7 min to obtain half of maximum bonding strength (760 g/cm2). The bonding strength of this glue to collagen-based casings increased with increasing of HSA concentration.The bonding strength of CAD-A glue increased with increasing CAD concentration up to 200 mM, and then decreased with increasing CAD concentration under the fixed HSA concentration (50 w/w%). The CAD-A glue showed excellent wound closure ability rather than fibrin glue when applied to the mouse skin. These results suggested that this developed glue had both tissue compatibility and bonding strength for use in clinical field.

In this study, three kinds of photo-reactive polymers, namely positively charged azidophenyl-derived polyallylamine (AzPhPAAm), negatively charged azidophenyl-derived poly(acrylic acid) (AzPhPAAc) and neutral azidophenyl-derived poly(ethylene glycol) (AzPhPEG), were synthesized by introduction of photo-reactive group (-N3) into polyallylamine, poly(acrylic acid) and poly(ethylene glycol), respectively. The structure of the azidophenyl-derived polymers was confirmed by 1H-NMR measurement. All of the photo-reactive polymers can be pattern-grafted on the surface of cells culture plate, which was confirmed by the optical microscopy observation. The contact angle decreased after surface modification by the photo-reactive polymers. Human mesenchymal stem cells (MSC) cultured on the modified surfaces showed different morphology. The cells adhered and spread more on the PAAm- and PAAc-grafted surfaces than on the PEG-grafted surface. The pellets formed on PAAm- and PEG-grafted surfaces in condrogenic differentiation medium were positively stained by safranin O/ fast green. But the pellet formed on PAAc-grafted surface did not show obviously positive staining for safranin O/ fast green. These results indicate that the PAAm- and PEG-grafted surfaces promoted chondrogenic differentiation of MSC.

A novel collagen sponge that can protect cell leakage during cell seeding was developed by wrapping all the surfaces except the upside of a collagen sponge with membrane that has pores smaller than cell. The collagen sponge was used for three-dimensional culture of human bone marrow-derived mesenchymal stem cells (MSCs). The cells adhered to the collagen, and proliferated to fill the spaces in the sponge. The cell seeding efficiency was higher than 95%. The MSCs cultured in the collagen sponge in the chondrogenic induction medium supplemented with TGF-β3 and BMP6 expressed genes encoding type II collagen, SOX9 and aggrecan. HE staining indicated the round morphology of differentiated cells and the extracelluler matrices were positively stained by safranin O and toluidine blue. Type II collagen and cartilage proteoglycan were detected by immunostaining with anti-type II collagen and anti-cartilage proteoglycan. These results suggest the chondrogenic differentiation of MSCs. The collagen sponge facilitated cell seeding and chondrogenic differentiation of MSCs, and will be useful for cartilage tissue engineering.

Polypyridine grafted with poly (ethylene glycol) copolymers (Py-g-PEG) with varying composition were prepared through radical copolymerization of methyl-terminated PEG macromonomer and 4-pyridylmethyl methacrylate, and the obtained copolymer spontaneously adsorbs from aqueous solution onto gold surfaces, where the pyridine parts act as the multipoint anchor to the surface and the PEG parts provide the strong steric repulsion between the chains. As a result, the highly protein repellent and stable surface was constructed through multipoint pyridine attachment as compared with singlepoint pyridine attachment. Furthermore, the property of multimolecular micellization for the obtained copolymer in aqueous milieu was examined to estimate the relation between the surface properties on the substrate and the self-assembling ability in aqueous milieu. Py-g-PEGs thus prepared are promising materials to functionalize metal and semiconductor materials and to self-assemble into micelles in biotechnological and biomedical fields.

Canine bone marrow stromal cells were cultured in a PLGA–collagen hybrid mesh in osteogenic medium in vitro and laminated to construct an osteo layer. Canine articular chondrocytes were cultured in the hybrid mesh in DMEM containing 10% FBS and laminated to construct a chondral layer. The osteo and chondral layers were sutured together and implanted subcutaneously in nude mice. The original round disc shape of the osteochondral constructs was preserved during the implantation. The osteo and chondral layers appeared red and glistening white, respectively. Histological examination of the implant specimens indicated that stromal cells and chondrocytes were evenly distributed throughout the scaffold. The laminated meshes were bound together and the two layers had a distinct interface between them. The cells showed a round morphology in the chondral layer and a spindle morphology in the osteo layer. In the chondral layer, spherical chondrocytes were surrounded by an abundant cartilaginous extracellular matrix. The round morphology and positive stain by safranin- O and toluidine blue, together with the expression of genes encoding type II collagen and aggrecan suggested the formation of neocartilage in the chondral layer. Expressions of genes encoding type I collagen and osteocalcin were detected in the osteochondral implant. These results indicate the formation of osteochondral-like tissue, and the hybrid mesh and lamination method may be useful for osteochondral tissue engineering.

Three-dimensional porous scaffolds play an important role for tissue engineering. The recent developments of porous scaffolds and their preparation methods, especially those developed by our group, are summarized in this review. A method for the preparation of biodegradable porous scaffolds has been developed by using pre-prepared ice particulates as porogen material. A kind of composite biodegradable porous scaffolds has been developed by forming collagen microsponges in the pores or interstices of a synthetic polymer sponge or mesh. A composite sponge of synthetic polymer, collagen and hydroxyapatite has been developed for hard tissue engineering. Bovine articular cartilage-like tissue has been engineered by culturing chondrocytes in the PLGA-collagen scaffolds.

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.

Recently, many kinds of methodology to regenerate articular cartilage have been developed. It is important to assess whether reconstructed cartilaginous tissue have appropriate molecular-structure for articular cartilage. There are numerous studies about biochemical and biomechanical properties of reconstructed cartilaginous tissue. However the noninvasive or nondestructive assessment of molecular-structure have not been performed on regenerated cartilage. In this study, we established the methodology of nondestructive molecular-structure assessment of regenerated cartilage using NMR spectroscopy, and performed the assessment on regenerated cartilaginous tissue prepared by chondrocyte-agarose culture model. As the results, the 1 H-NMR spectrum of the regenerated cartilaginous tissue showed peaks dominated similarly to chondroitin sulfate solution. In conclusion, this methodology using NMR spectroscopy could be useful for the assessment of regenerated cartilage.

A new method for the preparation of biodegradable porous scaffolds has been developed by using preprepared ice particulates as porogen material. A novel kind of hybrid biodegradable porous scaffold has been developed by forming collagen microsponges in the pores or interstices of a synthetic polymer sponge or mesh. A hybrid sponge of synthetic polymer, collagen and hydroxyapatite has been developed for hard tissue engineering. Bovine articular cartilage-like tissue has been engineered by culturing chondrocytes in the PLGA-collagen scaffolds.

Polyamides containing amino acid residues were synthesized through a chemoenzymatic route. At first, monomers containing amino acid esters at both ends were prepared from amino acid esters and bifunctional acid chlorides through a Scotten-Baumann reaction under organic condition. Diamine was used as bifunctional nucleophilic reagent to aminolysis the amino acid ester bonds of these monomers under protease catalysts, leading to the polycondensation to form the desired polyamides containing amino acid residues. Spectra characterizations confirmed the structure of obtained polyamides. The factors that influence the polymerization, including the type of enzymes, the amount of enzymes, and the water contents in the solvent, were also discussed. It is expected that the obtained polyamides have the potential for biomaterial applications.

A thin biodegradable hybrid mesh of synthetic poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen was used for three-dimensional culture of human skin fibroblasts. The hybrid mesh was constructed by forming web-like collagen microsponges in the openings of a PLGA knitted mesh. The behaviors of the fibroblasts on the hybrid mesh and PLGA knitted mesh were compared. The efficiency of cell seeding was much higher and the cells grew more quickly in the hybrid mesh than in the PLGA mesh. The fibroblasts in the PLGA mesh grew from the peripheral PLGA fibers toward the centers of the openings, while those in the hybrid mesh also grew from the collagen microsponges in the openings of the mesh resulting in a more homogenous growth. The proliferated cells and secreted extracellular matrices were more uniformly distributed in the hybrid mesh than in the PLGA mesh. Histological staining of in vitro cultured fibroblast/mesh implants indicated that the fibroblasts were distributed throughout the hybrid mesh and formed a uniform layer of dermal tissue having almost the same thickness as that of the hybrid mesh. However, the tissue formed in the PLGA mesh was thick adjacent to the PLGA fibers and thin in the center of the openings. Fibroblasts cultured in the hybrid mesh were implanted in the back of nude mouse. Dermal tissues were formed after 2 weeks and became epithelialized after 4 weeks. The results indicate that the web-like collagen microsponges formed in the openings of the PLGA knitted mesh increased the efficiency of cell seeding, improved cell distribution, and therefore facilitated rapid formation of dermal tissue having a uniform thickness. PLGA-collagen hybrid mesh may be useful for skin tissue engineering.

A new route for preparing hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) bioceramic has been described. An amorphous, nanosized, and carbonate-containing calcium phosphate powder that had a Ca:P ratio of 1.67 was synthesized from calcium diethoxide and phosphoric acid in ethanol via a sol-gel method. The powder was pressed at 98 MPa into green specimens and then heated to a temperature range of 500°-1300°C. At 600°C, the powder crystallized to a carbonated hydroxyapatite and a trace of β-tricalcium phosphate before converting to hydroxyapatite at 900°C. The thermal crystallization was associated with grain growth, shrinkage, and active surface diffusion. The activation energy of grain growth was 37 ± 2 kJ/mol. After sintering at 1100°C, the decomposition of carbonated hydroxyapatite generated a microporous ceramic with an average pore size of 0.2 μm and an open porosity of 15.5%. This microporous bioceramic can be used as a bone filler.

Canine anterior cruciate ligament (ACL) cells were isolated from the anterior cruciate ligament and subcultured in DMEM containing 10% FBS. After being subcultured twice, the ACL cells were seeded in a PLGA-collagen hybrid mesh and cultured in DMEM containing 10% FBS in vitro for 16 days. After 1 day, the cells adhered and spread well on collagen microsponges of the hybrid mesh. The cells proliferated and excreted extracellular matrices with culture time. After 16 days of culture, the cells and extracellular matrices occupied the spaces in the scaffold. At that point, the cell/mesh constructs were rolled up and implanted subcutaneously in the dorsum of nude mouse. The implants were harvested after 2, 4, 8, and 12 weeks of implantation and examined by histological staining. The implants were macroscopically white. Histological examination of cross sections of the implant by hematoxylin and eosin staining showed a large population of cells and newly deposited collagen in the rolled implants seeded with ACL cells. The cells were surrounded with bundles of collagen fibers. The layers in the rolled implant became integrated after 8 weeks. The polymer fibers could still be seen 8 weeks after implantation and gradually degraded and eventually disappeared after 12 weeks.

The enzymatic polycondensation of polyamides containing amino acid residues was demonstrated. The diamide monomers, containing amino acid (Phe, Leu, Val) esters at both ends, were synthesized using a Scotten–Baumann reaction. Polymerization was conducted through an enzymatic aminolysis of the ester bond by aliphatic diamine in organic solvents. Several proteases were employed to evaluate the enzyme specificity on the polymerization. α-Chymotrypsin was found to have the most effective catalyst activity on the monomer containing phenylalanine, reflecting the specific enzymatic process. Approximate molecular weight at about 7000 was obtained through this enzymatic polycondensation. The structures of the obtained polymers were confirmed employing IR and NMR spectra. The polymerization occurred in various solvents, including two- and one-phase systems. It is found that water content in organic solvent affects the yield.

Tissue engineering has been rapidly developing as an alternative approach to the repair and regeneration of damaged human tissue. A temporary scaffold is necessary to accommodate new cells and to guide the formation of tissue. Cell adhesion to a scaffold is the first step of cell behavior when it comes in contact with the scaffold surface and is an important consideration in tissue engineering. Bone marrow cells, chondrocytes, ligament cells and synovial cells were cultured in a PLGA–collagen hybrid mesh and their adhesion to the scaffold was studied. All four kinds of cells adhered to the hybrid mesh. The cells did not spread and showed a round morphology after culture for 30 min either in serum or in a serum-free medium. If cells were seeded at a high concentration, more cells were observed in the hybrid mesh. After culture for 6 h, the cells spread and showed a flatter morphology. The cells cultured in the serum medium spread out more than did those in serum-free medium. Calculations of cell seeding efficiencies were based on the number of cells that adhered to the mesh after culture for 30 min and for 6 h. The number of adhered cells increased with an increase of culture time for all four types of cells. For the mesenchymal stem cells, the medium composition and cell concentration affected seeding efficiency during the initial period. Cell seeding efficiency increased with an increase in cell concentration and the serum medium promoted cell adhesion, but not after culture for 6 h. The adhesion of the chondrocytes and ligament cells showed the same trend in that the efficiency of cell growth did not change in serum or serum-free medium, or in high or low cell concentration. For the synovial cells, cell concentration did not affect cell seeding efficiency, but more cells adhered to the hybrid mesh in the serum-free medium than did in the serum medium. These results suggest that the seeding conditions should be specified according the specific cell type when the PLGA–collagen hybrid mesh is used for tissue engineering.

Recently, an approach to restore cartilage defects, culturing autologous chondrocytes in vitro to create a three-dimensional tissue and then implanting the cultured tissue, has been developed. In this kind of approach, it is important to study whether the extracellular matrixes synthesized by cultured chondrocytes have appropriate mechanical property. In this study, we attempted to evaluate in detail mechanical properties of the extracellular matrix synthesized by chondrocytes in vitro. Therefore, we cultured bovine chondrocytes in agarose gel and assessed the changes in not only static but also dynamic mechanical properties during long-term culture. In addition, to assess the mechanical property of the extracellular matrix synthesized by chondrocytes only, we calculated the volume ratio of the extracellular matrix in agarose gel from the cross section and estimated the static Young's modulus of the extracellular matrix synthesized by cultured chondrocytes based on the Rule of Mixture . As a result, both the equilibrium aggregate modulus and dynamic Young's modulus of chondrocyte/agarose disks increased with time. The value of calculated modulus was larger than that of the pericellular matrix of cartilage explant. In conclusion, the biomechanical property of chondrocyte/agarose constructs and the matrix synthesized by chondrocytes were assessed in detail.

Canine chondrocytes isolated from articular cartilage were cultured in a PLGA-collagen hybrid mesh in DMEM containing 10% FBS in vitro for 10 days, and subsequently implanted subcutaneously in the dorsum of nude mouse. The formation of neo-cartilage tissue was examined. HE staining showed a spherical morphology of chondrocytes in the center of the explants and a fibroblast-like morphology in the periphery of the explants. The safranin O/fast green staining showed that the central part was positively stained for GAG while the periphery was negatively stained. The toluidine blue staining showed a similar result. The histological results demonstrated spherical chondrocytes surrounded by abundant cartilaginous matrices in the center of the explants. The fibro-tissue in the periphery became denser and the central cartilage tissue became thinner as the implantation period increased, suggesting the effects such as invasion of host fibroblasts and diffusion of soluble factors on the degeneration of newly formed cartilage tissue. Gene expression study using reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the expressions of type II collagen and aggrecan were detected and up-regulated, while that for type I collagen was down-regulated when the cells were cultured in the hybrid mesh. These results suggest the formation of articular cartilage in the subcutaneous dorsum of nude mouse.

Bovine chondrocytes were isolated from the shoulder and knee articular joints of a calf, seeded in biodegradable porous polymer scaffolds, and implanted subcutaneously in the dorsa of athymic nude mice to tissue engineer articular cartilage in vivo. Hybrid sponge of poly l -lactic acid (PLLA) and collagen was used as the porous scaffold with PLLA sponge as the control. Chondrocytes were seeded in the sponges at a density of 5×107 cells/ml. PLLA–collagen hybrid sponge was easily seeded, but PLLA sponge required prewetting of the sponge before cell seeding. The scaffold-cell constructs were cultured in vitro for 1 week and implanted subcutaneously in the dorsa of athymic nude mice. The implants were harvested after in vivo incubation for 2, 4 and 8 weeks. The PLLA–collagen hybrid sponge implants maintained their original shapes, as did the PLLA sponge. The implants were examined histochemically by Hematoxylin/eosin and Safranin O/fast green staining, and immunohistochemically by anti-bovine type II collagen antibody. The presence of cartilaginous extracellular matrices indicated by positive staining of Safranin O and anti-bovine type II collagen antibody increased, while neovascularization decreased with an increased implantation period. Cartilaginous tissue was more widely formed and more homogeneously distributed in the PLLA–collagen hybrid sponge than in the PLLA sponge throughout implantation period. Hybridization of PLLA sponge and collagen sponge facilitated cell seeding, prevented the scaffold from collapsing and promoted the formation of cartilaginous tissue in vivo.

Cryopreserved human bone marrow-derived mesenchymal cells (hBMCs) were thawed and replated in culture dishes. Viability of the cells immediately after thawing was 98%. When the cells were cultured in the presence or absence of dexamethasone, the cells proliferated to reach confluency at culture day 7. The cells treated with dexamethasone formed abundant mineralized nodules at their extracellular regions after about culture day 14, while the cultures without dexamethasone did not exhibit bone formation. The calcein uptake into the extracellular matrix layer was detected in the dexamethasone-treated cells with the increase in fluorescent intensity with culture periods but not in the nontreated cells. These results suggest that human bone marrow-derived mesenchymal cells retain bone-forming capability even after cryopreservation/thawing.

The rotation culture method adopted in our experiments induced numerous aggregates of bone marrow stromal cells (BMSCs); this method augments the frequency of cell–cell contacts and thereby facilitates cell aggregation. Cell condensation plays an important role in differentiation and development. Cell adhesion molecules and the extracellular matrix (ECM) are important regulators of cell adhesion and apoptosis. Therefore, we investigated not only whether aggregates of BMSCs could be formed by rotation culture, but also whether expression of N-cadherin and β1-integrin was involved in the formation of cell aggregates and in the avoidance of apoptosis. The culture medium was supplemented with fibroblast growth factor-2 (FGF-2) or transforming growth factor-β1 (TGF-β1), expected to prompt the formation of cell aggregates. This study enabled the formation of numerous aggregates of BMSCs by rotation culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that FGF-2 and TGF-β1 increased expression of N-cadherin and β1-integrin. Morphological analysis by light microscopy indicated that FGF-2 and TGF-β1 increased cell–cell adhesion and formation of cell aggregates. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay indicated that FGF-2 and TGF-β1 prevented apoptosis. These results suggest that numerous aggregates of BMSCs were formed efficiently by rotation culture since FGF-2 and TGF-β1 increase cell–cell adhesion and rotation increases cell–cell contact.