Takaaki Arahira

Effects of proliferation and differentiation of mesenchymal stem cells on compressive mechanical behavior of collagen/β-TCP composite scaffold.

The primary aim of this study is to characterize the effects of cell culture on the compressive mechanical behavior of the collagen/β-tricalcium phosphate (TCP) composite scaffold. The composite and pure collagen scaffolds were fabricated by the solid-liquid phase separation technique and the subsequent freeze-drying method. Rat bone marrow mesenchymal stem cells (rMSCs) were then cultured in these scaffolds up to 28 days. Compression test of the scaffolds with rMSCs were conducted periodically. Biological properties such as cell number, alkaline phosphatase (ALP) activity, and gene expressions of osteogenetic bone markers were evaluated during cell culture. The microstructural changes in the scaffolds during cell culture were also examined using a scanning electron microscope. The compressive elastic modulus was then correlated with those of the biological properties and microstructures to understand the mechanism of variational behavior of the macroscopic elastic property. The composite scaffold exhibited higher ALP activity and more active generation of osteoblastic markers than the collagen scaffold, indicating that β-TCP can activate the differentiation of rMSCs into osteoblasts and extracellular matrix (ECM) formation such as type I collagen and the following mineralization. The variational behavior of the compressive modulus of the composite scaffold was affected by both the material degradation and the proliferation of cells and the ECM formation. In the first stage, the modulus of the composite scaffold tended to increase due to cell proliferation and the following formation of network structure. In the second stage, the modulus tended to decrease because the material degradation such as ductile deformation of collagen and decomposition of β-TCP were more effective on the property than the ECM formation. In the third stage, active calcification by formation and growth of mineralized nodules resulted in the recovery of modulus. It is concluded that the introduction of β-TCP powder into the porous collagen matrix is very effective to improve the mechanical and biological properties of collagen scaffold prepared for bone tissue engineering. Furthermore, the compressive modulus of the composite scaffold is strongly affected by the material degradation and the ECM formation by stem cells under in vitro culture condition.

Variation of mechanical behavior of β-TCP/collagen two phase composite scaffold with mesenchymal stem cell in vitro

The primary aim of this study is to characterize the variational behavior of the compressive mechanical property of bioceramic-based scaffolds using stem cells during the cell culture period. β-Tricalcium phosphate (TCP)/collagen two phase composites and β-TCP scaffolds were fabricated using the polyurethane template technique and a subsequent freeze-drying method. Rat bone-marrow mesenchymal stem cells (rMSCs) were then cultured in these scaffolds for up to 28 days. Compression tests of the scaffolds with rMSCs were periodically conducted. Biological properties, such as the cell number, alkaline phosphatase (ALP) activity, and gene expressions of osteogenesis, were evaluated. The microstructural change due to cell growth and the formation of extracellular matrices was examined using a field-emission scanning electron microscope. The compressive property was then correlated with the biological properties and microstructures to understand the mechanism of the variational behavior of the macroscopic mechanical property. The porous collagen structure in the β-TCP scaffold effectively improved the structural stability of the composite scaffold, whereas the β-TCP scaffold exhibited structural instability with the collapse of the porous structure when immersed in a culture medium. The β-TCP/collagen composite scaffold exhibited higher ALP activity and more active generation of osteoblastic markers than the β-TCP scaffold.

Development and characterization of hybrid tubular structure of PLCL porous scaffold with hMSCs/ECs cell sheet

Tissue engineering offers an alternate approach to providing vascular graft with potential to grow similar with native tissue by seeding autologous cells into biodegradable scaffold. In this study, we developed a combining technique by layering a sheet of cells onto a porous tubular scaffold. The cell sheet prepared from co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs) were able to infiltrate through porous structure of the tubular poly (lactide-co-caprolactone) (PLCL) scaffold and further proliferated on luminal wall within a week of culture. Moreover, the co-culture cell sheet within the tubular scaffold has demonstrated a faster proliferation rate than the monoculture cell sheet composed of MSCs only. We also found that the co-culture cell sheet expressed a strong angiogenic marker, including vascular endothelial growth factor (VEGF) and its receptor (VEGFR), as compared with the monoculture cell sheet within 2 weeks of culture, indicating that the co-culture system could induce differentiation into endothelial cell lineage. This combined technique would provide cellularization and maturation of vascular construct in relatively short period with a strong expression of angiogenic properties.Graphical abstract

Characterization of Tensile Mechanical Behavior of MSCs/PLCL Hybrid Layered Sheet

A layered construct was developed by combining a porous polymer sheet and a cell sheet as a tissue engineered vascular patch. The primary objective of this study is to investigate the influence of mesenchymal stem cells (MSCs) sheet on the tensile mechanical properties of porous poly-(l-lactide-co-ε-caprolactone) (PLCL) sheet. The porous PLCL sheet was fabricated by the solid-liquid phase separation method and the following freeze-drying method. The MSCs sheet, prepared by the temperature-responsive dish, was then layered on the top of the PLCL sheet and cultured for 2 weeks. During the in vitro study, cellular properties such as cell infiltration, spreading and proliferation were evaluated. Tensile test of the layered construct was performed periodically to characterize the tensile mechanical behavior. The tensile properties were then correlated with the cellular properties to understand the effect of MSCs sheet on the variation of the mechanical behavior during the in vitro study. It was found that MSCs from the cell sheet were able to migrate into the PLCL sheet and actively proliferated into the porous structure then formed a new layer of MSCs on the opposite surface of the PLCL sheet. Mechanical evaluation revealed that the PLCL sheet with MSCs showed enhancement of tensile strength and strain energy density at the first week of culture which is characterized as the effect of MSCs proliferation and its infiltration into the porous structure of the PLCL sheet. New technique was presented to develop tissue engineered patch by combining MSCs sheet and porous PLCL sheet, and it is expected that the layered patch may prolong biomechanical stability when implanted in vivo.

Effects of osteoblast-like cell seeding on the mechanical properties of porous composite scaffolds

In tissue engineering technology, polymer–ceramics or polymer–polymer composites have been considered as advanced scaffolds having mechanical stability, biocompatibility, cell proliferation, and easy processability. However, the relationship between the mechanical properties and the cell proliferation behavior of such composite scaffolds has not been clarified yet. In this study, two types of composite scaffolds, poly(ethylene terephthalate) (PET) fiber/collagen and β-tricalcium phosphate (β-TCP)/gelatin scaffolds, were investigated. MC3T3-E1 cells were cultured in these scaffolds under appropriate conditions. Compression tests were then periodically conducted to evaluate the compressive elastic modulus. It was found that the modulus of the scaffolds containing cells increased with the cell culture period. It is noted that the modulus of the β-TCP/gelatin with cells was approximately seven times larger than that of the PET fiber/collagen with cells.

Characterization and in vitro evaluation of biphasic α-tricalcium phosphate/β-tricalcium phosphate cement

Biphasic calcium phosphate consisting of hydroxyapatite (HA) and β-tricalcium phosphate(β-TCP) is an excellent bone substitute with controllable bioresorbability. Fabrication of biphasic calcium phosphate with self-setting ability is expected to enhance its potential application as bone substitute. In this study, mixtures of α-TCP and β-TCP with various compositions were prepared through α-β phase transition of α-TCP powder at 1000°C for various periods. These powders were mixed with 0.25M Na2HPO4 at a P/L ratio of 2, and then hardened at 37°C at 100% RH for up to 24h. Material properties of biphasic HA/β-TCP cement with different α-TCP/β-TCP composition were characterized. These cements were also evaluated with respect to cell response in vitro using MC3T3-E1 cell lines. In conclusion, mechanical and biological properties of HA/β-TCP cement could be controlled by changing the heat treatment time of α-TCP powder at 1000°C. In vitro results indicated that cell proliferation and ALP activity increased with increase β-TCP content.

Tissue Reaction to a Novel Bone Substitute Material Fabricated With Biodegradable Polymer-calcium Phosphate Nanoparticle Composite

Purpose:The aim of this study was to evaluate the effectiveness of a novel bone substitute material fabricated using a biodegradable polymer-calcium phosphate nanoparticle composite. Methods:Porous structured poly-L-lactic acid (PLLA) and hydroxyapatite (HA) nanoparticle composite, which was fabricated using solid-liquid phase separation and freeze-drying methods, was grafted into bone defects created in rat calvarium or tibia. Rats were killed 4 weeks after surgery, and histological analyses were performed to evaluate new bone formation. Results:Scanning electron microscopic observation showed the interconnecting pores within the material and the pore diameter was approximately 100 to 300 &mgr;m. HA nanoparticles were observed to be embedded into the PLLA beams. In the calvarial implantation model, abundant blood vessels and fibroblastic cells were observed penetrating into pores, and in the tibia model, newly formed bone was present around and within the composite. Conclusions:The PLLA-HA nanoparticle composite bone substitute developed in this study showed biocompatibility, elasticity, and operability and thus has potential as a novel bone substitute.

An Evolutionary Rule Mining Method for Continuous Value Prediction from Incomplete Database and Its Application Utilizing Artificial Missing Values

A rule mining method for continuous value prediction has been proposed to handle incomplete databases using a graph structure-based evolutionary computation technique. The method extracts the associative local distribution rule, the consequent part of which has a narrow distribution of continuous variables. A set of associative local distribution rules was applied for continuous value prediction. Instances including missing values were predicted using the predictor. A method for constructing a probability distribution of predicted values for each focusing instance was considered based on extracted rule sets. The proposed method offers some flexibility by allowing users to define the conditions of prediction rules. The method can quit rule extraction when a sufficient number of rules are extracted for building a predictor. Therefore, it is suitable for prediction when large datasets are involved. In addition, we have proposed an application of artificial missing values to improve the effectiveness of the developed rule-based prediction system. Artificial missing values are applied to avoid the sharp boundary problem encountered when discretizing continuous variables. Attribute values near the boundary in discretization are treated as missing values. The performance of the artificial missing value-based prediction method was evaluated, and the results showed that the proposed method was effective for prediction.

Development and characterization of bioceramic/polymer composite scaffold with drug release function for bone tissue engineering

Hydroxyapatite porous material was coated by bioabsorbable polymers such as PCL or PLGA containing statin which is a commercial drug known to enhance bone formation in vivo condition. The compressive mechanical properties of the composite scaffolds were evaluated to assess the effects of polymer coating. Drug release tests were also performed to assess the effects of coating polymer and coating method on the drug release rate. It is concluded that the stain/PLGA(PCL)/HA scaffolds can be one of the potential candidates for bone tissue engineering.

Development and characterization of CNT/biopolymer electrodes for bio-fuel cell

CNT/biopolymer composite materials having continuous porous structure were developed as the electrode materials for bio-fuel cell. Biodegradable polymer, PLLA, and natural biopolymer, collagen, were utilized as the polymer matrix of the composite materials. Enzymes were also distributed in the composites. Microstructures of the composites were observed using a field-emission scanning electron microscope. A simple experimental set-ups of bio-fuel cell were also constructed using the composite materials as the electrodes. Fundamental electric property such as electrification was then examined using the systems.