[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM.
[show abstract][hide abstract] ABSTRACT: Funnel-like sponges of collagen incorporated with glycosaminoglycan (GAG) were prepared by freeze-drying using ice particulates as templates. The funnel-like collagen-GAG sponges showed similar porous structures to those of funnel-like collagen sponges. The funnel-like collagen-GAG and collagen sponges have one top surface layer and one bulk porous layer. The large, top surface pores were determined by ice particulates that were used as templates, and the inner bulk pores were determined by freezing temperature. The funnel-like pore structures facilitated homogenous cell distribution, improved cell viability, and resulted in homogenous tissue formation. Incorporation of GAG increased the mechanical property and cell viability of collagen sponges.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Bioactive and Compatible Polymers 07/2010; 25:360-373. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties. To reconstruct cartilage tissue, articular chondrocytes from bovine humeral heads were embedded in agarose gel and cultured in vitro for up to 4 weeks. The nFCD of the cartilage was determined using the MRI gadolinium exclusion method. The equilibrium modulus was determined using a compressive stress relaxation test, and the dynamic modulus was determined by a dynamic compression test. The equilibrium compressive modulus and dynamic modulus of the tissue-engineered cartilage increased with an increase in culture time. The nFCD value--as determined with the [Gd-DTPA(2-)] measurement using the MRI technique--increased with culture time. In the regression analysis, nFCD showed significant correlations with equilibrium compressive modulus and dynamic modulus. From these results, gadolinium-enhanced MRI measurements can serve as a useful predictor of the biomechanical properties of tissue-engineered cartilage.
Journal of Biomechanical Engineering 07/2010; 132(7):071014. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: A novel method to fabricate highly interconnected porous hyaluronic acid (HA) scaffolds with open surface pore structures was developed by using embossed ice particulates as a template. HA sponges were cross-linked by water-soluble carbodiimide (WSC) and the optimal cross-linking condition was analyzed by infrared spectroscopy. Cross-linking with 50 mM WSC in a 90% (v/v) ethanol/water solvent mixture assured the highest degree of cross-linking and most stable structure and, therefore, was used to cross-link the HA sponges. Observation with a scanning electron microscope showed that the HA scaffolds had funnel-like porous structures. There were large, open pores on the top surfaces and inner bulk pores under the top surface of the funnel-like HA sponges. The inner bulk pores were interconnected with the large, top surface pores and extended into the whole sponge. The pore morphology and density of the large, top surface pores were dependent on the dimension and density of the ice particulates. The size of the inner bulk pores was dependent on the freezing temperature. The funnel-like pore structures of the HA sponges facilitated cell penetration into the inner pores of the sponges and resulted in homogenous cell distribution in the sponges.
[show abstract][hide abstract] ABSTRACT: Development of porous scaffolds with open surface pore structures is required for tissue engineering to deliver cells into the three-dimensional spaces in the scaffolds and improve cell distribution. This study demonstrated a new type of funnel-like chitosan sponge prepared using ice particulates as a template. The funnel-like chitosan sponges had a hierarchical bilayer porous structure of a surface layer and an interconnected bulk porous layer. The top surface porous layer consisted mainly of large open pores. The bulk porous layer was beneath the large surface pores and consisted of small pores that were connected with the large surface pores. The large surface pores were dependent on the shape, dimension, and density of the embossing ice particulates, while the bulk pores were dependent on the freezing temperature. The large open surface pores and interconnected bulk pores in the funnel-like chitosan sponges facilitated cell seeding and cell distribution from the surface into the inner bulk pores. Cells cultured in the funnel-like chitosan sponges showed high viability, high proliferation, and homogenous tissue formation. Such funnel-like chitosan sponges will be useful for tissue engineering.
Journal of Biomedical Materials Research Part B Applied Biomaterials 05/2010; 93(2):341-50. · 2.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: To realize real-time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate-type viscometer, combined with an upright epi-fluorescence microscope. The apparatus allowed real-time evaluation of platelet-material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 microL) under shear flow conditions. To study the dynamic behavior of platelet-material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small-scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large-scale flow system. Therefore, the newly developed cone and plate-type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion.
[show abstract][hide abstract] ABSTRACT: Chondrocytes change their morphology and dedifferentiate to fibroblast-like cell during in vitro expansion culture. The phenotype variation may have some effect on the mechanical properties of living chondrocytes. This study was performed to determine the effect of the living chondrocytes’ passage number on their stiffness using atomic force microscopy (AFM). We used freshly isolated (P0) and serially passaged (P1-P5) bovine articular chondrocytes (BACs) and compared the results of Young's modulus measurements with changes in the morphology of the cells. We found that the BACs phenotype variation was accompanied with a decrease in the elastic modulus of the living cells. The change in stiffness was most rapid for the first two passages (P0–P2). The comparison of the mechanical properties of chondrocytes with intact cellular membranes and cells with removed membranes enabled us to conclude that the cellular membrane can play a significant role in the mechanical properties of chondrocytes.
[show abstract][hide abstract] ABSTRACT: The effects of hardystonite (Ca(2)ZnSi(2)O(7), CSZn) and tricalcium phosphate (beta-TCP) on the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) were compared by directly culturing MSCs on ceramic disks (contact mode) or separately culturing cells with ceramic disks (non-contact mode). In non-contact mode, the CSZn ceramic supported MSC proliferation more strongly than did the beta-TCP ceramic. However, in contact mode, the MSCs proliferated more quickly on the beta-TCP ceramic than they did on the CSZn ceramic. Alkaline phosphatase (ALP) staining and osteogenic gene expression analysis showed that the CSZn and beta-TCP ceramics had significant effects on the promotion of the osteogenic differentiation of MSCs in both non-contact and contact mode. Furthermore, in contact mode, the CSZn disk promoted the osteogenic differentiation of MSCs more strongly than did the beta-TCP disks. Even without the induction of dexamethasone and beta-glycerophosphate, CSZn stimulated the osteogenic differentiation of MSCs. These results suggest that CSZn ceramic would be a useful candidate material for bone regeneration and hard tissue engineering.
Journal of Biomaterials Applications 10/2009; 25(1):39-56. · 2.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: An extracellular microenvironment, including an extracellular matrix (ECM), is an important factor in regulating stem cell differentiation. During tissue development, the ECM is dynamically remodeled to regulate stem cell functions. Here, we developed matrices mimicking ECM remodeling during the osteogenesis of mesenchymal stem cells (MSCs). The matrices were prepared from cultured MSCs controlled at different stages of osteogenesis and referred to as "stepwise osteogenesis-mimicking matrices." The matrices supported the adhesion and proliferation of MSCs and showed different effects on the osteogenesis of MSCs. On the matrices mimicking the early stage of osteogenesis (early stage matrices), the osteogenesis occurred more rapidly than did that on the matrices mimicking undifferentiated stem cells (stem cell matrices) and the late stage of osteogenesis (late stage matrices). RUNX2 was similarly expressed when MSCs were cultured on both the early stage and late stage matrices but decreased on the stem cell matrices. PPARG expression in the MSCs cultured on the late stage matrices was higher than for those cultured on the stem cell and early stage matrices. This increase of PPARG expression was caused by the suppression of the amount of beta-catenin and downstream signal transduction. These results demonstrate that the osteogenesis-mimicking matrices had different effects on the osteogenesis of MSCs, and the early stage matrices provided a favorable microenvironment for the osteogenesis.
Journal of Biological Chemistry 09/2009; 284(45):31164-73. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Tissue Engineering Part C Methods 08/2009; 16(3):329-38. · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three-dimensional (3D) porous scaffolds constructed from biodegradable synthetic polymers are frequently used in tissue engineering. Their surfaces are hydrophobic and require treatment to be changed to hydrophilic before use in cell culture. We developed a novel surface modification for 3D porous scaffolds made of synthetic polymers by coating the surfaces of the pores with a nanothick collagen layer. First, a collagen aqueous solution was introduced under reduced pressure to fully fill the pores of the PLGA sponges. The collagen-containing sponges were then centrifuged to remove any excess collagen solution. Finally, the sponges were freeze-dried to form a thin collagen layer. Scanning electron microscopy observation and water absorption tests demonstrated that the excess collagen was removed; the effect of modification was evident when the collagen-containing sponges were centrifuged at high centrifugal acceleration. Scanning probe microscopy analysis demonstrated the formation of a nanometer-thick collagen layer on the PLGA surface. The collagen-coated PLGA sponges facilitated cell seeding and spatial distribution. The method will be useful for the surface modification of 3D porous scaffolds.
Journal of Biomedical Materials Research Part B Applied Biomaterials 06/2009; 90(2):864-72. · 2.31 Impact Factor