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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.

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.

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．

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Biodegradable polymers have been adopted as materials of three-dimensional (3D) scaffolds for regenerating cartilage-like tissues by using tissue engineering. On the other hand, it is well recognized that phenotypic control of seeded chondrocytes is indispensable for constructing not fibrous but hyaline cartilage-like tissue. According to the context, type II collagen is focused as a candidate of scaffold materials for articular cartilage regeneration. This study compared viability and gene expressions of seeded chondrocytes in type I and type II collagen sponges. The results show that the seeded chondrocytes were well distributed and alive in the both sponges in 20 days of culture, and no significant difference in type I, type II and aggrecan gene expressions was found between both sponges. The effect of type II collagen as a scaffold material having outside in-signals on regeneration of hyaline cartilage-like tissue was not detected at least at the initial stage of tissue regeneration.

Topical hyperbaric oxygen (topical HBO), used for moderate acute and chronic open wounds, is one of several strategies utilized to accelerate the healing process. We used topical HBO with a polyethylene bag filled with oxygen and monitored the oxygen concentration in the bag during the treatment. We also tried to treat two cases of severe refractory foot ulcer with this method, as an alternative to flap surgery or amputation. The result was that the oxygen concentration level has became 90% after 15 min and the level was kept over 90% after this time. The infection healed within 3 weeks and the ulcers were closed in both cases successfully. If topical HBO can be enhanced, the epithelialization may be beneficial not only as a useful treatment for severe ulcers, but also as a device in the field of tissue engineering.

Recently, hydroxyapatite/collagen (HAp/Col) nanocomposites incorporated into bone remodeling processes have been developed. Because bone tissue reaction to the composites is obviously suitable for bone tissue engineering, a novel HAp/Col–alginate hybrid material, as one variety of the HAp/Col implants, was prepared to expand the limited applications of the composite. The ability of HAp/Col–alginate as a bone filler was investigated in the rat femur and as a carrier of BMP using an ectopic bone formation model. Active bone formation around the material and tissue invasion into the material were observed throughout the experiment in bone, compared to those observed for simple alginate and porous HA. Furthermore, HAp/Col–alginate was investigated as a carrier of recombinant human bone morphogenetic protein 2 (rh-BMP2). The HAp/Col–alginate (20 μl) with the rh-BMP2 (100 μg/ml, 15 μl) showed bone formation throughout the implant 5 weeks after implantation without obvious deformation of the material, whereas bone formation was observed only in a part of a squashed collagen sponge.

One of the major problems in autologous chondrocyte transplantation for cartilage defect repair is the acquisition of a sufficient number of chondrocytes. Chondrocytes lose their cartilaginous phenotype during expansion in monolayer culture. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into chondrocytes under appropriate conditions. In order to explore the feasibility of applying these cells simultaneously for cartilage tissue engineering, human MSC and bovine mature articular chondrocytes (BCH) were mixed and cocultured. Mixed pellet culture showed upregulation of cell proliferation, cartilaginous extracellular matrix production and type II collagen gene expression. MSCs mediated a paracrine effect on BCH through growth factor secretion or cell-to-cell interaction, upregulating the cartilaginous phenotype of BCH.

We developed a new tissue-engineered bone graft model composed of bovine bone marrow stromal cells (BMSCs), and beta tricalcium phosphate (beta TCP) porous beads. Such cell–bead sheets were prepared by means of cultivation of BMSCs with the beads in alpha-MEM culture medium supplemented with 10% fetal bovine serum. The shape of the sheets was possible to change depending on that of a plastic mold having a circle or quadrangle shape. Addition of poundage on the overlaid cell–bead sheets induced the connection of these sheets with each other, and achievement of thicker implant formation. A nonwoven fabric sheet of poly- l -lactic acid (PLLA) could also be inserted in between each cell–bead sheet. Consequently, bone implants having a size of 20×20×5 mm were successfully developed. This kind of techniques would be useful in thick implants for other tissue implants.

Ligament injuries are one of most common injuries to the knee joint; however, little is known about how ligaments heal and what kinds of precursor cells participate in ligament repair. Ligament cells were thought to be members of one of the lineages from mesenchymal stem cells (MSCs). However, there have been no reports on the differentiation mechanism of MSC to ligament cells because they do not have known phenotypic markers. To identify the anterior cruciate ligament (ACL)-specific phenotypic marker, we screened a cDNA library derived from rabbit ACL cells, using the signal sequence trap by the retrovirus-mediated expression screening (SST-REX) method. We detected known clones representing 135 independent proteins. Among them, 120 were secreted proteins and 15 were membrane proteins. Some of the clones encoded proteins that have never been reported to be expressed in ACLs. Therefore, the data could provide a better understanding of ACL cells and could help in finding their phenotypic markers.

A biodegradable hybrid scaffold of synthetic polymer, poly (DL-lactic-co-glycolic acid) (PLGA), and naturally derived polymer, collagen, was prepared by forming collagen microsponges in the pores of PLGA sponge. This was then used as the three-dimensional scaffold for tissue engineering of bovine articular cartilage, both in vitro and in vivo. In vitro studies show that hybridization with collagen facilitated cell seeding in the sponge and raised seeding efficiency. Chondrocytes adhered to the collagen microsponges, where they proliferated and secreted extracellular matrices with time, filling the space within the sponge. Hematoxylin and eosin staining revealed that most of the chondrocytes after 4 weeks of culture, and almost all cell types after 6 weeks of culture, maintained their phenotypically rounded morphology. While new tissue formed, the scaffold degraded and lost almost 36.9% of its original weight after 10 weeks. Subcutaneous implantation studies in nude mice demonstrated more homogeneous tissue formation in hybrid sponge than in PLGA sponge. The new tissue formed maintained the original shape of the hybrid sponge. The synthetic PLGA sponge, serving as a skeleton, facilitated easy formation into desired shapes and provided appropriate mechanical strength to define the ultimate shape of engineered tissue. Incorporation of collagen microsponges facilitated cell seeding and homogeneous cell distribution and created a favorable environment for cellular differentiation. The hybrid sponge could therefore represent a promising candidate as a three-dimensional scaffold for articular cartilage tissue engineering.

Purpose: Tissue engineering of the urinary bladder using autologous cells and biodegradable scaffold is a promising method for augmentation. The authors developed 2 hybrid scaffolds by combining poly (DL-lactic-co-glycolic acid; PLGA) mesh for mechanical strength with collagen sponge or gel suitable for cell seeding. The aim of this study was to compare collagen as a scaffold between collagen sponge and gel and to construct a tissue-engineered urinary bladder wall utilizing these hybrid scaffolds. Methods: The PLGA mesh-collagen hybrid scaffolds were prepared by introducing collagen sponge or gel into the PLGA knitted mesh. Urothelial and smooth muscle cells were obtained from porcine urinary bladder wall and were cultured in their respective media. The cells were seeded on these hybrid scaffolds. These constructs were analyzed morphologically and immunohistochemically. Results: The urothelial layer was generated 3 dimensionally by culturing urothelial cells with PLGA mesh and collagen sponge. The smooth muscle layer was constructed by culturing smooth muscle cells with PLGA mesh and collagen gel. And a novel tissue-engineered urinary bladder wall was constructed laminating the urothelial and smooth muscle layers. Conclusions: Ex vivo construction of urinary bladder wall using hybrid scaffolds prepared by combining PLGA mesh with collagen sponge or gel was successful. This tissue-engineered urinary bladder wall allows easy handling and may become a promising tool for bladder augmentation.

Damaged articular cartilage has a very limited capability of self-healing. The need for improved treatment of cartilage defects, which involve over 1 percent of population in Japan, has motivated research to create regenerated cartilage. Regenerated cartilage is engineered by culturing autologous chondrocytes on biodegradable scaffolds. We studied changes in biomechanical property, especially dynamic visco-elasticity during 6 weeks of in vitro culture. The results suggest that the ability of water holding was increased by new extracellular matrix (ECM) synthesis during 4 weeks of in vitro culture, although the ability of solid matrix (ECM and scaffold) to resist compression elastically was reduced.

Hyperbaric oxygen (HBO) is a method of augmenting, intermittently, oxygen availability to tissues. We examined the effect of three different HBO exposures on the healing of experimentally induced ligament lacerations in the right hind limb of 44 male Wistar rats. Animals were divided into four groups after ligament injury: (a) control group, animals breathed room air at 1 ATA (atmosphere absolute) in a hyperbaric chamber for 60 min; (b) HBO treatment at 1.5 ATA for 30 min once a day, (c) HBO treatment at 2 ATA for 30 min once a day, (d) 2 ATA for 60 min once a day. At 14 days post-ligament injury, we compared the ligaments of the four treatment groups for gross appearance, histology and expression of pro-alpha(I) mRNA by northern hybridization. Our results indicate that HBO was effective in promoting ligament healing compared to control (p < 0.01). Of these three exposures, HBO at 2 ATA for 60 min was the most effective, resulting in enhanced extra-cellular matrix deposition as measured by collagen synthesis.

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β3 (TGF-β3) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β3 mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β3 mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.

Anterior cruciate ligament (ACL) is continuously exposed to tensile load, which is generally recognized to be an essential factor in maintaining the physiological homeostasis of the ACL fibers during daily activities. Although this kind of mechanical stimulation is clearly known to play an important role in quickening repair of ACL injury, responses of ACL cells to mechanical stimulation still remain unclear. We have investigated whether extracellular signal-regulated kinase 1/2 (ERK1/2) in human ruptured ACL-derived cells are phosphorylated and involved in type I collagen (Col I) and decorin gene expression by mechanical stimulation. Mechanical stretch stimulates time-dependent phosphorylation of ERK1/2 in human ruptured ACL-derived cells. While Col I gene expression levels were increased in 48-h continuous mechanical stretch, decorin gene expression levels were decreased for 24-h continuous stimulation. Decorin gene expression levels were increased, however, in mechanical stretch+PD98059, which is an inhibitor of ERK pathway, treated cells as compared with control cells at 24 h. Our results show that ERK pathway is stimulated by mechanical stretch, and may be involved in regulation of Col I and decorin gene expression in ACL-derived cells.

Bovine chondrocytes were isolated from the shoulder articular joints of a calf, seeded in biodegradable porous polymer scaffolds, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. Hybrid sponge of poly( dl -lactic-co-glycolic acid) (PLGA) and collagen was used as the porous scaffold with PLGA sponge and collagen sponge used as the controls. Chondrocytes were seeded in low (1×10 7 cells/ml) and high (5×10 7 cells/ml) densities. Before implantation, the scaffold-cell constructs were cultured in vitro for 1 week. The implants were harvested after in vivo incubation of 2, 4, 6, and 8 weeks. The PLGA–collagen hybrid sponge implants maintained their original shapes, as did the PLGA sponge, whereas the collagen sponge collapsed. The mechanically strong PLGA sponge functioned as a skeleton and prevented the embedded collagen sponge from collapsing. The implants were examined histochemically by haematoxylin and eosin staining, by safranin O/fast green staining, and immunohistochemically by anti-collagen type II antibody. The percent of cartilaginous extracellular matrices increased, while neovascularization decreased with an increased implantation period. A greater amount of homogeneous cartilaginous tissue formed when chondrocytes were seeded at a higher density. The morphology of chondrocytes, the expression of cartilaginous matrices in the PLGA–collagen hybrid sponge, was similar to those in collagen sponge. The cartilaginous matrices were more homogeneously distributed in the PLGA–collagen hybrid sponge and collagen sponge than in the PLGA sponge. Hybridization of the PLGA sponge and collagen sponge facilitated cell seeding and promoted the in vivo formation of cartilaginous tissue.

In order to elucidate signal transduction mechanism on how cells sense mechanical deformation, cytosolic Ca transients, by poking with a heat-blunted glass micropipette, were directly observed in human microvascular endothelial cell (HMEC). Cytosolic Ca transients were observed in almost all the HMECs poked with a micropipette. The transients started at the point of poking, and propagated at the peripheral regions of the poked cell. Those transients were also observed to propagate to the adjacent cells. Neither transient of poked cells nor propagation to adjacent cells was detected under the condition of depletion of extracellular Ca or intracellular Ca stores. Either stretch-activated channel or voltage-sensitive ion channel blockage inhibited significantly Ca transients. Phospholipase C inhibition, resulting in inhibition of inositol 1,4,5-triphosphate (IP 3 ) production, caused no Ca transient by poking. The results suggested that Ca influx might be a trigger for Ca release from intracellular Ca stores by IP 3 in the case of HMEC poking experiments.

Recently, synthetic porous hydroxyapatite (HA) has attracted a great deal of attention as a bone graft substitute in the field of tissue engineering. Combining with bone marrow-derived osteoblasts (BMO), bone formation has been confirmed in vivo and in vitro. However, it is still necessary to obtain more bone formation within porous HA for clinical usage, and, thus, new methods need to be developed. We hypothesized that low pressure during subculture would cause more osteoblastic cells to migrate into the pores of porous HA blocks, resulting in more bone tissue formation in vivo. In the present study, we examined six experimental groups with different pressures from 760 to 10 mm Hg applied to porous HA blocks loaded by bone marrow-derived osteoblasts. For in vivo testing, the 2-week subcultured HA/BMO composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested at 2, 4 or 8 weeks after implantation. Then, they were prepared for biochemical analysis of alkaline phosphatase (ALP) activity, bone osteocalcin (OCN) content and histological analysis. ALP activity and OCN content in the 100-mm Hg pressure group were highest among the different groups 4 and 8 weeks after implantation (P < 0.001). Light microscopy revealed mature bone formation in HA/BMO composite at 4 weeks after implantation. In the scanning electron microscopy (SEM) study, mineralized collagenous extracellular matrix as well as active osteoblasts was observed in HA/BMO composite at 2 weeks after implantation. We concluded that the application of low-pressure system to subculture of bone cells to porous HA blocks is beneficial to increase bone tissue formation in vivo.

This review article will present our work regarding osteopontin (OPN) function in bone remodeling and its application to tissue engineering in bone. OPN, one of the major non-collagenous bone proteins, has an important role in bone remodeling. Our study suggested that OPN acts not only as a trigger for osteoblast early differentiation but activates osteoclast resorption. On this point of view, we proposed a model of bone remodeling via integrin α V β 3 –OPN binding, in which OPN could act as a signal molecule between osteoblasts and osteoclasts in the process of remodeling. Moreover, OPN was shown to induce the in vivo osteogenesis of a bone marrow-derived osteoblast/porous hydroxyapatite (HA) constructs. Our application of adenovirus-mediated gene transfer of osteopontin to primary osteoblasts is also presented.

To accelerate wound healing after implantation of tissue-engineered skin products, higher density inoculation of fibroblasts would be important. Therefore, we attempted to develop a tissue-engineered skin with a higher density of fibroblasts by formation of cell aggregates combined with a biodegradable polymer. Our method consisted of rotationally shaking with nontreated dishes, declining fibroblast-material interactions, and augmenting cell–cell interaction. Under the culture condition, normal neonatal human dermal fibroblasts formed spheroidal aggregates. The formation of the fibroblast-aggregates was accelerated by the addition of insulin, dexamethasone, basic fibroblast growth factor, and ascorbic acid to basal medium, and by the control of shaking time. In fibroblast-aggregates formed under a basal medium, the level of gene expression related to wound healing was higher than that of a monolayer when one added the hormones and growth factors. Therefore, tissue-engineered skin products with aggregates and scaffolds would become an artificial skin with potentiality of higher wound healing.