Naoki Kawazoe

The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering.

3D biodegradable porous scaffold plays a very important role in articular cartilage tissue engineering. We developed hybrid structures of 3D scaffolds that combined the advantages of natural type I collagen and synthetic PLGA knitted mesh. The mechanically strong PLGA mesh served as a skeleton while the collagen microsponges facilitated cell seeding and tissue formation. The scaffolds were divided into 3 groups: (1) THIN: collagen microsponge formed in interstices of PLGA mesh; (2) SEMI: collagen microsponge formed on one side of PLGA mesh; (3) SANDWICH: collagen sponge formed on both sides of PLGA mesh. Bovine chondrocytes were cultured in these scaffolds and transplanted subcutaneously into nude mice for 2, 4, and 8 weeks. All three groups of transplants showed homogeneous cell distribution, natural chondrocyte morphology, and abundant cartilaginous ECM deposition. Production of GAGs per DNA and the expression of type II collagen and aggrecan mRNA were much higher in the SEMI and SANDWICH groups than in the THIN group. When compared to native articular cartilage, the mechanical strength of the engineered cartilage reached 54.8%, 49.3% in Youngs modulus and 68.8%, 62.7% in stiffness, respectively, in SEMI and SANDWICH. These scaffolds could be used for the tissue engineering of articular cartilage with adjustable thickness. The design of the hybrid structures provides a strategy for the preparation of 3D porous scaffolds.

Decellularized matrices for tissue engineering

Importance of the field: Biomimetic scaffolds and substrates of extracellular matrices (ECMs) play an important role in the regulation of cell function and in the guidance of new tissue regeneration, as an ECM has the intrinsic cues necessary to communicate with and dictate to cells. Areas covered in this review: This paper reviews the latest developments in ECM scaffolds and substrates obtained from decellularized tissues, organs or cultured cells and their application in tissue engineering. The ECM composition, structure, interaction with surrounding cells, preparation method and usage in the regeneration of various tissues and organs are summarised. What the reader will gain: The advantages and challenges of decellularized matrices are highlighted. Take home message: Similarity in the composition, microstructure and biomechanical properties of the decellularized scaffolds and substrates to those of the native tissues and organs maximizes the promotion effect in the regeneration of both structural and functional tissues and organs. Simple tissues as well as complicated organs have been decellularized and decellularization methods have been optimized to completely remove the cellular components while keeping the ECM intact.

Cultured cell-derived extracellular matrix scaffolds for tissue engineering.

Cell-derived extracellular matrix (ECM) scaffolds have received considerable interest for tissue engineering applications. In this study, ECM scaffolds derived from mesenchymal stem cell (MSC), chondrocyte, and fibroblast were prepared by culturing cells in a selectively removable poly(lactic-co-glycolic acid) (PLGA) template. These three types of ECM scaffolds were used for in vitro cultures of MSC and fibroblasts to examine their potential as scaffolds for cartilage and skin tissue engineering. The MSC were cultured in MSC- and chondrocyte-derived ECM scaffolds. The ECM scaffolds supported cell adhesion, promoted both cell proliferation and the production of ECM and demonstrated a stronger stimulatory effect on the chondrogenesis of MSC compared with a conventional pellet culture method. Histological and immunohistochemical staining indicated that cartilage-like tissues were regenerated after the MSC were cultured in ECM scaffolds. Fibroblasts were cultured in the fibroblast-derived ECM scaffolds. Fibroblasts proliferated and produced ECM to fill the pores and spaces in the scaffold. After 2 weeks of culture, a uniform multilayered tissue was generated with homogenously distributed fibroblasts. Cell-derived ECM scaffolds have been demonstrated to facilitate tissue regeneration and will be a useful tool for tissue engineering.

Pore size effect of collagen scaffolds on cartilage regeneration

Scaffold pore size is an important factor affecting tissue regeneration efficiency. The effect of pore size on cartilage tissue regeneration was compared by using four types of collagen porous scaffolds with different pore sizes. The collagen porous scaffolds were prepared by using pre-prepared ice particulates that had diameters of 150-250, 250-355, 355-425 and 425-500μm. All the scaffolds had spherical large pores with good interconnectivity and high porosity that facilitated cell seeding and spatial cell distribution. Chondrocytes adhered to the walls of the spherical pores and showed a homogeneous distribution throughout the scaffolds. The in vivo implantation results indicated that the pore size did not exhibit any obvious effect on cell proliferation but exhibited different effects on cartilage regeneration. The collagen porous scaffolds prepared with ice particulates 150-250μm in size best promoted the expression and production of type II collagen and aggrecan, increasing the formation and the mechanical properties of the cartilage.

Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering

Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of synthetic and natural extracellular matrix components. In this study, nanofibrous scaffolds were obtained by electrospinning a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type-I collagen in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HIFP). The resulting fibers ranged from 300 to 600 nm in diameter. Their surfaces were characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis and atomic force microscopy. The PHBV and collagen components of the PHBV/collagen nanofibrous scaffold were biodegraded by PHB depolymerase and a type-I collagenase aqueous solution, respectively. The cell culture experiments indicated that the PHBV/collagen nanofibrous scaffold accelerated the adhesion and growth of NIH3T3 cells more effectively than the PHBV nanofibrous scaffold, thus making the former a good scaffold for tissue engineering.

Development of Stepwise Osteogenesis-mimicking Matrices for the Regulation of Mesenchymal Stem Cell Functions

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 β-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.

In vitro evaluation of biodegradation of poly(lactic-co-glycolic acid) sponges.

Evaluation of the degradability of porous scaffolds is very important for tissue engineering. A protocol in which the condition is close to the in vivo pH environment was established for in vitro evaluation of biodegradable porous scaffolds. Degradation of PLGA sponges in phosphate-buffered solution (PBS) was evaluated with the protocol. The PLGA sponges degraded with incubation time. For the first 12 weeks, the weight loss increased gradually and then remarkably after 12 weeks. In contrast, the number-average molecular weight (Mn) decreased dramatically for the first 12 weeks and then less markedly after 12 weeks. Thermal analysis showed that the glass transition temperatures (Tg) decreased rapidly for the first 12 weeks, and the change became less evident after 12 weeks. These results suggest that the degradation mechanism of PLGA sponges was dominated by autocatalyzed bulk degradation for the first 12 weeks and then by surface degradation after 12 weeks. Physical aging was observed during incubation at 37 degrees C. The heterogeneous structure caused by physical aging might be one of the driving forces that induced autocatalyzed bulk degradation. The degradation mechanism was further supported by the data of pH change and the morphology of the degraded PLGA sponges. The autocatalyzed acidic products flooded out after 8 weeks, the pH dropped, and the walls of the sponges became more porous. The increase of the pore surface area facilitated surface degradation after 12 weeks. The pH was in the range between 7.43 and 7.24 during the entire incubation time. The protocol suppressed extreme changes of the pH and will be useful in the biodegradation evaluation of porous scaffolds for tissue engineering.

Cartilage tissue engineering using funnel-like collagen sponges prepared with embossing ice particulate templates.

Three-dimensional porous scaffolds of collagen have been widely used for tissue engineering and regenerative medicine. In this study, we fabricated funnel-like collagen sponges with open surface pore structures by a freeze-drying method that used embossing ice particulates as a template. By controlling the size of the ice particulates and the temperature of freezing, collagen sponges with different pore structures were prepared. To investigate the effects of different pore structures on cartilage regeneration, the funnel-like collagen sponges were used to culture bovine articular chondrocytes. Scaffolds that were prepared with 400microm ice particulate templates and a freezing temperature of -3 degrees C resulted in the best cell distribution, ECM production, and chondrogenesis. Although funnel-like collagen sponges prepared with 400microm ice particulate templates and a freezing temperature of -1 degrees C and 720microm ice particulates and a freezing temperature of -3 degrees C, showed even cell distribution, the cell seeding efficiencies and sGAG amount per cell were low. However, the scaffolds prepared with 400microm ice particulate templates and a freezing temperature of -5 degrees C or -10 degrees C showed a limited effect on the improvement of cell distribution and chondrogenesis. Control collagen sponges without ice particulates failed to support the formation of homogenous cartilage-like tissue. These results indicate that funnel-like collagen sponges were superior to control collagen sponges and that scaffolds prepared with 400microm ice particulate templates at -3 degrees C were optimal for cartilage tissue engineering.

Adipogenic differentiation of individual mesenchymal stem cell on different geometric micropatterns.

Micropatterned surfaces are very useful to control cell microenvironment and investigate the physical effects on cell function. In this study, poly(vinyl alcohol) (PVA) micropatterns on polystyrene cell-culture plates were prepared using UV photolithography. Cell adhesive polystyrene geometries of triangle, square, pentagon, hexagon, and circle were surrounded by cell nonadhesive PVA to manipulate cell shapes. These different geometries had the same small surface areas for cell spreading. Human mesenchymal stem cells (MSCs) were cultured on the micropatterned surface, and the effect of cell geometry on adipogenic differentiation was investigated. MSCs adhered to the geometric micropatterns and formed arrays of single cell with different shapes. The distribution patterns of actin filaments were similar among these cell shapes and remolded during adipogenesis. The adipogenic differentiation potential of MSCs was similar on the small size triangular, square, pentagonal, hexagonal, and circular geometries according to lipid vacuoles staining. This simple micropatterning technique using photoreactive molecules will be useful for creating micropatterns of arbitrary design on an organic surface, and cell functions can be directly and systematically compared on a single surface without external factors resulting from separate cell culture and coating method.

Spatial immobilization of bone morphogenetic protein-4 in a collagen-PLGA hybrid scaffold for enhanced osteoinductivity

The introduction of bioactive molecules into three-dimensional porous scaffolds to mimic the in vivo microenvironment is a promising strategy for tissue engineering and stem cell research. In this study, bone morphogenetic protein-4 (BMP4) was spatially immobilized in a collagen-PLGA hybrid scaffold with a fusion BMP4 composed of an additional collagen-binding domain derived from fibronectin (CBD-BMP4). CBD-BMP4 bound to the collagen-PLGA hybrid scaffold and the BMP4-immobilized hybrid scaffold supported cell adhesion and proliferation. The osteogenic induction effect of the immobilized CBD-BMP4 was investigated with three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the BMP4-immobilized collagen-PLGA hybrid scaffold. The in vivo implantation experiment demonstrated that the immobilized CBD-BMP4 maintained its osteoinductive activity, being capable of up-regulating osteogenic gene expression and biomineralization. The strong osteoinductivity of the BMP4-immobilized scaffold suggests it should be useful for bone tissue engineering, stem cell function manipulation and bone substitutes.