Key Engineering Materials Vols. 342-343

Abstract: Previously, we have developed a novel method for suspension culture of anchoragedependent animal cells using biodegradable polymer nanospheres. In this study, we compared the polymer nanosphere culture method to dextran microcarrier culture method, which is a conventional suspension culture method. Most of human dermal fibroblasts (91 ± 5 %) cultured with polymer nanospheres formed aggregates on day 2. Most of cells (92 ± 7 %) attached onto microcarriers by 4 h. Microcarrier culture method had a lower apoptotic activity (3.4 folds on day 4), compared to the nanosphere culture. The microcarrier culture method had a higher cell growth (2.4-fold versus 1.7- fold growth on day 4) than the nanosphere culture. Although the polymer nanosphere culture method did not yield better outcomes than the microcarrier culture, the polymer nanosphere culture method may offer advantages over the microcarrier culture method with respect to cell protection from the shear stress during agitation at high speed and cell transplantation without enzyme digestion process to harvest cultured cells.

Abstract: The purpose of this study was to evaluate the bone interface response to different surface using bone remodeling rate (BRR) under cyclic loading. The tibiae of 10 New Zealand white rabbits were used. Each tibia of rabbits received 2 implants, one with anodic oxidation surface and the other with machined surface. Dynamic loading, a cyclic load of 100 N with 100 cycles was applied for 2 weeks. All implants were divided into 4 groups according to surface treatment and loading; (1) machined surface, unloaded, (2) anodic oxidation surface, unloaded, (3) machined surface, loaded, and (4) anodic oxidation surface, loaded. To determine the rate of bone formation, a series of fluorochrome bone labeling materials (tetracycline, alizarin red, and calcein green) were administrated intramuscularly at 10, 11, 12 weeks. To study the formation time of new bone, ground sections were examined under confocal laser scanning microscopy. Resonance frequency analysis (RFA) values were measured at the time of first surgery, second surgery and sacrifice. From this study, it was concluded that implants with anodic oxidation surface had higher value than machined surface on RFA measurement and anodic oxidation surface has more resistance to loading than machined surface.

Abstract: Mesenchymal stem cells(MSCs) have the potential to self-replicate, proliferate and differentiate into chondrocytes, adipocytes, osteocytes and myocytes. Recent studies indicate that vitamin K2 plays a role in bone metabolism. But the mechanism is still unclear. We evaluated effect of vitamin K2 on osteogenesis and adipogenesis of MSCs from umbilical cord blood(UCB). By exposing MSCs to osteogenic and adipogenic medium with and without vitamin K2, the effects of vitamin K2 were analyzed. The results showed that vitamin K2 inhibits adipogenesis while at the same time it stimulates osteoblastic differentiation. These data are expected to provide novel information needed for successful therapeutic development for various types of osteoporosis and similar diseases related to bone formation.

Abstract: Alginate, which is isolate from brown seaweed, a bioabsorbable long chain polysaccharides of guluronic acid and mannuronic acid. The authors produced alginate, fibroblastgrowth- factor mixed alginate and alginate-collagen complex as a disc form. For study of wound healing, full thickness skin defects were made on the dorsal area of the animal model. And then alginate, fibroblast-growth-factor mixed alginate, alginate-collagen complex, vaseline gauze as control were applied on the wound and evaluated grossly and histopathologically. For biocompatibility test, alginate and alginate-collagen complex discs were implanted on back of New Zealand white rabbits. And we measured size of wound healing, size of wound epithelization, size of wound contracture at 5, 10, 15, 20, and 25 days. Four weeks after implantation, implanted animals were examined immunologically against alginate and collagen. Alginate and FGF-mixed alginate, alginate-collagen complex group showed statistically higher percentage of wound contraction and wound healed than control group(p<0.05) Alginate-collagen complex group and FGF-mixed alginate group showed statistically higher percentage of wound healed than alginate group. In conclusion, high-purified alginate from seaweeds by our purification method, has the ability of wound healing and addition of FGF or collagen increase the ability of alginate, it shows the possibility of alginate as dermal substrate.

Abstract: In this study, various dental stem cells were isolated and their mineralization ability was evaluated. First, we isolated postnatal stem cells from human dental tissues such as dental pulp (DPSCs), periodontal ligament (PDLSCs), periapical follicle (PAFSCs) and mandibular bone marrow (MBMSCs). Then, we cultured these dental stem cells in the mineralization condition for 21 days. Ability of these stem cells to mineralize was evaluated by Alizarin Red staining, quantitative analysis of Alizarin Red, alkaline phosphatase (ALP) activity, and RT-PCR. The calcium deposits of PAFSCs were sparsely scattered throughout the adherent layer, whereas MBMSCs and PDLSCs cultures produced extensive sheets of calcified deposits over the entire adherent layer. Quantitative Alizarin Red staining and the ALP activity also were high in MBMSCs and PDLSCs. In the osteogenic differentiation condition, all of dental stem cells showed upregulated expression of the mineralization-related gene. Our findings suggest that isolated DPSCs, PDLSCs, PAFSCs and MBMSCs have the different mineralization abilities.

Abstract: The objective of this study was to quantify the zonal difference of the in situ chondron’s Poisson effect under different magnitudes of compression. Fluorescence immunolabeling for type VI collagen was used to identify the pericellular matrix (PCM) and chondron, and a series of fluorescent confocal images were recorded and reconstructed to form quantitative three-dimensional models. The zonal variations in the mechanical response of the chondron do not appear to be due to zonal differences in PCM properties, but rather seem to result from significant inhomogeneities in relative stiffnesses of the extracellular matrix (ECM) and PCM with depth.

Abstract: Bone marrow derived-stromal cells (BMSCs) are pluriplotent progenitors for a variety of cell types, including osteoblast, chondrocyte, adipocyte, and so on. Stem cell research has enormous potential in the future clinical treatment of a wide range of diseases. Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. However, we don’t have exact evaluation methods and safeguards for clinical application. In this study, we investigated tracking BMSCs differentiation, localization, toxicity, and migration in vivo. The fluorescent vector used in our studies did not affect BMSCs viability or their ability to undergo osteogenic and adipogenic differentiation in vitro. During differentiation, EGFP-BMSCs by Oil Red O and Alizarin Red S were stained. EGFP-BMSCs were transplanted into the femoral region in autologous rabbit. After one month, these cells were detectable by confocal microscopy and RTPCR. Transplanted EGFP-BMSCs were not detected another organs (spleen, kidney, liver, and muscle) in immunohistochemistry and RT-PCR. In organ-function test and cell-toxicity examination, there is no difference between the before and after EGFP-BMSCs transplantation. we observed that transplanted EGFP-BMSCs were not affected cell-toxicity and migration. This results offer some evaluation methods and safeguards for clinical application using BMSCs. In further study, it will be needed to test reproductive and developmental toxicity after transplantation and observe migration of EGFP-BMSCs after transplantation.

Abstract: Although tissue engineering is an area with great potential, it still has few applications due to the lack of biocompatible, biodegradable materials with suitable mechanical properties. Recently, several biodegradable materials were developed, of which poly(propylene fumarate (PPF) is one of the most notable. It degrades into fumaric acid and propylene glycol, which are both biocompatible products. Microstereolithography is a new technology that can be used to fabricate free-form 3-D microstructures by dividing a desired shape into many slices of a given horizontal thickness. This technology requires a low-viscosity resin to fabricate fine structures. However, the viscosity of PPF is too high to fabricate 3D structures using microstereolithography. Therefore, we reduced the viscosity of the resin by adding diethyl fumarate (DEF). Then, we added a photoinitiator to photo-crosslink the DEF/PPF resin, and fabricated 2.5-D scaffolds using our system. We confirmed that microstereolithography technology is effective in scaffold fabrication. The fabricated 2.5-D scaffolds were seeded with fibroblasts and the cells attached well after seeding.

Abstract: Poly-L-lactic acid (PLLA) is a desirable and very attractive polymer for fabricating porous scaffolds. As of now, a solvent casting method with organic solvents has been used in scaffold fabrication process. However, residual organic solvents in the scaffolds have the problems of decreasing the effect of osteogenic induction due to the hindrance of bioceramic by polymer solution and it’s harmfulness in vivo. To avoid these disadvantages of scaffolds by organic solvent casting method, we developed a new method fabricating polymer (PLLA)/ceramic (β -TCP) composite scaffolds by baking method without using solvent, and then we tested properties of scaffolds on animals. As the result, non-toxicity has been proved through animal experiment and newly fabricated polymer/ceramic composites by a novel sintering method were induced rapid bone regeneration through enhancing the interaction of cells and a bone induction factor without any host immune response.

Abstract: Two passaged (P2) immature porcine articular chondrocytes were used to fabricate an engineered cartilage tissue in an in vitro scaffold-free system with or without insulin like growth factor 1 (IGF-1). This study shows the possibility of the fabrication of structurally regular neocartilage tissue using passaged chondrocytes in the scaffold-free system with insulin like growth factor-1(IGF-1).