Fibril matrices created with collagen from the marine fish barramundi for use in conventional three-dimensional cell culture.

Collagen obtained from fish offal (skin, scales, and bones) is required from some religious and ethnic groups, thus indirectly increasing demands for fish collagen for biomedical applications. The limitation of fish collagen is its lower thermal stability compared to mammalian collagen. In this study, we focused on collagen extracted from scales of the marine fish barramundi (Lates calcarifer) and demonstrated the suitability for the collagen to be utilized in collagen fibril matrices (CFM). Collagen was extracted from the scales through pepsin-digestion and purified (designated as "BC"). The denaturation temperature (Td) for BC was determined to be 36.4 °C, one of the highest among fish collagens. BC formed CFM which was thermally stable at 37 °C, while Td was lower than 37 °C. This could be explained by the fast fibril formation, initiating at temperatures near 20 °C in a temperature-elevated process. As a result, the NIH3T3 cells were successfully encapsulated in the CFM of BC and cultured three-dimensionally for 7 d. The cells spread and exhibited well-developed pseudopodia in the CFM of BC as observed in the CFM of pig collagen matrices. This is the first report on fish CFM used for conventional 3-D cell culture.

Crosslinking Efficacy and Cytotoxicity of Genipin and Its Activated Form Prepared by Warming It in a Phosphate Buffer: A Comparative Study.

The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1-5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

In situ gelation properties of a collagen-genipin sol with a potential for the treatment of gastrointestinal ulcers.

We investigated the potential of collagen-genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20 min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2-1 mM, warranting the use of in situ gelling collagen-genipin sols for endoscopic treatments of gastrointestinal ulcers.

In Vitro Parallel Evaluation of Antibacterial Activity and Cytotoxicity of Commercially Available Silver-Containing Wound Dressings.

PURPOSE: This study evaluated the in vitro antibacterial activity and cytotoxicity of various commercially available silver-containing dressings (Ag dressing). METHODS: Biohesive Ag (hydrocolloid, silver sulfadiazine), Aquacel® Ag (nonwoven fabric, ionic silver [Ag]), Algisite™ Ag (nonwoven fabric, Ag), Mepilex® Ag (foam, silver sulfate), and PolyMem® Ag (foam, nanocrystalline silver) were tested for characteristics of Ag release, antibacterial activity, and cytotoxicity. The release of Ag was investigated in cell culture medium at immersion periods of 6, 24, and 48 hours. The antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa were accessed by a disc diffusion test. The cytotoxicity was evaluated using V79 cells, by an extraction method. RESULTS: The cytotoxicity was not a monotonic function of the antibacterial activity among the Ag dressings and could not be simply explained by Ag-release properties. Biohesive Ag was regarded as a slow-release Ag dressing, showing the lowest cytotoxicity, while the antibacterial activity was classified as "strong" or "significant" against the two species of bacteria. Aquacel Ag and Algisite Ag showed higher antibacterial activity and cytotoxic effects, which were supported by the higher Ag release. Mepilex Ag showed the highest release of Ag, and the cytotoxicity was the highest among the Ag dressings. However, the antibacterial activity was classified as "significant" or "no activity" for P. aeruginosa and S. aureus, respectively. PolyMem Ag showed the lowest Ag release, and the antibacterial activity classified as "significant" or "no activity" for S. aureus and P. aeruginosa, respectively, whereas the cytotoxicity was similar to those of Aquacel Ag and Algisite Ag. CONCLUSION: The efficacy and adverse effects of the Ag dressings revealed differences that should be considered by clinicians during wound management.

Evaluation of gelatin hydrogel as a potential carrier for cell transportation.

We prepared uncleaved gelatin composed mainly of collagen α-, ß-, and γ-chains. Gelation and melting of uncleaved gelatin occurred rapidly with moderate decrease and increase in temperature (23°C-37°C). The viability of cells encapsulated in the gelatin gel was greater than 96% after 7 d at 23°C.

Temperature-responsive gelation of type I collagen solutions involving fibril formation and genipin crosslinking as a potential injectable hydrogel.

We investigated the temperature-responsive gelation of collagen/genipin solutions using pepsin-solubilized collagen (PSC) and acid-solubilized collagen (ASC) as substrates. Gelation occurred in the PSC/genipin solutions at genipin concentrations 0-2 mM under moderate change in temperature from 25 to 37°C. The PSC/genipin solutions exhibited fluidity at room temperature for at least 30 min, whereas the ASC/genipin solutions rapidly reached gel points. In specific cases PSC would be preferred over ASC as an injectable gel system. The temperature-responsive gelation of PSC/genipin solutions was due to temperature responses to genipin crosslinking and collagen fibril formation. The elastic modulus of the 0.5% PSC/genipin gel system could be adjusted in a range of 2.5 to 50 kPa by the PSC and genipin concentrations, suggesting that a PSC/genipin solution is a potential injectable gel system for drug and cell carriers, with mechanical properties matching those of living tissues.

Fabrication of high-density collagen fibril matrix gels by renaturation of triple-helix collagen from gelatin.

Collagen-based 3-D hydrogels often lack sufficient mechanical strength for tissue engineering. We developed a method for fabrication of high-density collagen fibril matrix (CFM) gels from concentrated solutions of uncleaved gelatin (UCG). Denatured random-coil UCG exhibited more rapid and efficient renaturation into collagen triple-helix than cleaved gelatin (CG) over a broad range of setting temperatures. The UCG solution formed opaque gels with high-density reconstituted collagen fibrils at 28-32 °C and transparent gels similar to CG at <25 °C. The unique gelation properties of UCG enabled the encapsulation of cultured cells in CFM of high solid volume (>5%) and elasticity (1.28 ± 0.15 kPa at 5% and 4.82 ± 0.38 kPa at 8%) with minimal cell loss. The elastic modulus of these gels was higher than that of conventional CFM containing 0.5% collagen. High-strength CFM may provide more durable hydrogels for tissue engineering and regenerative medicine.

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits.

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i-QDs) in the reparative tissues, following the allogeneic transplantation of three-dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein-70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i-QDs were added to the culture medium for 24 h. Scaffold-free cartilaginous aggregates formed from i-QD-labelled MSCs (i-MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin-O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti-mortalin antibody-conjugated i-QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i-MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects.

Stable and nondisruptive in vitro/in vivo labeling of mesenchymal stem cells by internalizing quantum dots.

Progress in stem cell research has prioritized the refinement of cell-labeling techniques for in vitro and in vivo basic and therapeutic studies. Although quantum dots, because of their optical properties, are emerging as favorable nanoparticles for bioimaging, substantial refinements or modifications that would improve their biocompatibility are still required. We report here that internalizing quantum dots (i-QDs) generated by their conjugation with an internalizing antibody against a heat shock protein-70 family stress chaperone, mortalin, offered an efficient, genetically noninvasive, nontoxic, and functionally inert way to label mesenchymal stem cells (MSCs). The i-QD-labeled MSCs underwent normal adipocyte, osteocyte, and chondrocyte differentiation in vitro and in vivo, suggesting the potential application of i-QDs in in vivo diagnostics, regenerative and therapeutic medicine.

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Gene expression analysis of major lineage-defining factors in human bone marrow cells: effect of aging, gender, and age-related disorders.

Adult bone marrow cells (BMCs) include two populations:;mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat; and hematopoietic stem cells (HSCs), which produce all mature blood lineage. To study the effect of aging, gender, and age-related disorders on lineage differentiation, we performed quantitative RT-PCR to examine mRNA expression of the major factors defining BMC lineage, cbfa1 for osteoblasts, ppar-gamma for adipocytes, sox9 for chondrocytes, and rankl for osteoclasts, in bone marrow from 80 healthy subjects and patients (14-79 years old) with two age-related disorders: osteoarthritis (OA) and rheumatoid arthritis (RA). Two apoptosis-related genes, bcl-2 and drak1, were studied. RANKL and PPAR-Gamma levels exhibited a clear positive correlation with age in female patients, but not in males, with a slight age-related decline in CBFa1 transcripts. DRAK1 expression showed an age-associated ascending trend with significantly greater transcripts of RANKL and DRAK1 in females (p < 0.01). Compared with age-matched controls, RA patients exhibited increased RANKL, PPAR-Gamma, and DRAK1 mRNA levels (p < 0.05), and OA showed the higher RANKL and PPAR-Gamma transcripts (p < 0.05). Furthermore, SOX9 and DRAK1 expressions in the RA group were higher than in the OA group (p < 0.05). Our data indicate that aging and age-related disorders affect gene expressions differently, suggesting that in aging, the lineage of bone marrow cells was modified with prominent changes in decreased bone marrow osteoblastogenesis, increased adipogenesis and osteoclastogenesis, while in age-related disorders, marrow adipogenesis and the activity or number of osteoclasts may play an important role in the pathogenesis of arthritic bone loss.

Repair of large osteochondral defects with allogeneic cartilaginous aggregates formed from bone marrow-derived cells using RWV bioreactor.

Our objective was to examine the technique of regenerating cartilage tissue from bone marrow-derived cells by three-dimensional (3D) culture using the rotating wall vessel (RWV) bioreactor. Three-dimensional and cylindrical aggregates of allogeneic cartilage with dimensions of 10 x 5 mm (height x diameter) formed by the RWV bioreactor were transplanted into osteochondral defects of Japanese white rabbits (Group T, n = 15). For the control, some osteochondral defects were left empty (Group C, n = 18). At 4, 8, and 12 weeks postimplantation, the reparative tissues were evaluated macroscopically, histologically, and biochemically. In Group T at as early as 4 weeks, histological observation, especially via safranin-O staining, suggested that the reparative tissues resembled hyaline cartilage. And we observed no fibrous tissues between reparative tissue and adjacent normal tissues. In the deeper portion of the bony compartment, the osseous tissues were well remodeled. At 4 and 8 weeks postimplantation, the mean histological score of Group T was significantly better than that of Group C (p < 0.05). The glycosaminoglycans (GAG)/DNA ratio in both groups increased gradually from 4 to 8 weeks and then decreased from 8 to 12 weeks. We herein report the first successful regeneration of cartilage in osteochondral defects in vivo using allogeneic cartilaginous aggregates derived from bone marrow-derived cells by 3D culture using the RWV bioreactor.

Cartilaginous tissue formation from bone marrow cells using rotating wall vessel (RWV) bioreactor.

This is the first successful report of the rapid regeneration of three-dimensional large and homogeneous cartilaginous tissue from rabbit bone marrow cells without a scaffold using a rotating wall vessel (RWV) bioreactor, which simulates a microgravity environment for cells. Bone marrow cells cultured for 3 weeks in DMEM were resuspended and cultured for 4 weeks in the chondrogenic medium within the vessel. Large cylindrical cartilaginous tissue with dimensions of (1.25 +/- 0.06) x (0.60 +/- 0.08) cm (height x diameter) formed. Their cartilage marker expression was confirmed by mRNA expressions of aggrecan, collagen type I and II, and glycosaminoglycan (GAG)/DNA ratio. Their cartilaginous properties were demonstrated by toluidine blue, safranin-O staining, and polarization.

Nanopillar sheets as a new type of cell culture dish: detailed study of HeLa cells cultured on nanopillar sheets.

Nanopillar sheets were fabricated with high-aspect ratio structures with a diameter of 160-1,000 nm and a height of 1 mum by nanoimprinting. The suitability of nanopillar sheets as a new type of cell culture dish was examined by studying the behavior of HeLa cells cultured on the sheets using light microscopy, scanning electron microscopy, and fluorescence microscopy observing actin and vinculin molecules. The nanopillar structure enabled easy subculture of the cells from the sheets without conventional trypsinization. Moreover, the HeLa cells divided and proliferated on the sheets in a different way to that found on petri dish because of the manner in which the cells adhered to the materials.