Effects of culture conditions on the mechanical and biological properties of engineered cartilage constructed with collagen hybrid scaffold and human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) has been used as one of the new cell sources in osteochondral tissue engineering. It has been well known that control of their differentiation into chondrocytes plays a key role in developing engineered cartilages. Therefore, this study aims to develop a fundamental protocol to control the differentiation and proliferation of MSCs to construct an engineered cartilage. We compared the effects of three different culture conditions on cell proliferation, extracellular matrix formation and the mechanical response of engineered cartilage constructed using a collagen-based hybrid scaffold and human MSCs. The experimental results clearly showed that the combined culture condition of the chondrogenic differentiation culture and the chondrocyte growth culture exhibited statistically significant cell proliferation, ECM formation and stiffness responses as compared to the other two combinations. It is thus concluded that the combination of the differentiation culture with the subsequent growth culture is recommended as the culture condition for chondrogenic tissue engineering using hMSCs.

Characterization and thermal decomposition of synthetic carbonate apatite powders prepared using different alkali metal salts.

Two types of synthetic carbonate apatite [potassium-containing carbonate apatite (CAK) and sodium-containing carbonate apatite (CANa)] were prepared and characterized by thermogravimetric analysis, X-ray diffraction analysis (XRD) and Fourier transform infrared spectroscopy. The chemical formulas of carbonate apatite were determined to be Ca9.36K0.12(PO4)5.12(CO3)0.88(OH)1.73 and Ca8.72Na1.33(PO4)4.96(CO3)1.04(OH)1.80, respectively. Thermogravimetric analysis showed that the final weight loss at 1,200°C reached about 11.2% for CAK and 13.9% for CANa. Carbonate loss gradually occurred above 150°C and continued to 1,200°C. The crystallinity of the apatite phase was found to be much improved between 800 and 850°C for CAK and 750 and 800°C for CANa, where rapid carbonate loss occurred. A small amount of CaO formed above 900°C. For CANa, NaCaPO4 also formed above 700°C in both apatites. Although the lattice parameters of the carbonate apatites varied with temperature, the final a and c lattice parameters attained constant values of 0.9421 and 0.6881 nm.

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.

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

Biphasic calcium phosphate consisting of hydroxyapatite (HA) and ß-tricalcium phosphate(ß-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 ß-TCP with various compositions were prepared through α-ß 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/ß-TCP cement with different α-TCP/ß-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/ß-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 ß-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 µm. 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.

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.

Variation of mechanical behavior of ß-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. ß-Tricalcium phosphate (TCP)/collagen two phase composites and ß-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 ß-TCP scaffold effectively improved the structural stability of the composite scaffold, whereas the ß-TCP scaffold exhibited structural instability with the collapse of the porous structure when immersed in a culture medium. The ß-TCP/collagen composite scaffold exhibited higher ALP activity and more active generation of osteoblastic markers than the ß-TCP scaffold.

Effects of proliferation and differentiation of mesenchymal stem cells on compressive mechanical behavior of collagen/ß-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/ß-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 ß-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 ß-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 ß-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.

In vitro bone formation by mesenchymal stem cells with 3D collagen/ß-TCP composite scaffold.

Recent years, various kinds of natural polymers and bioceramics has been used to develop porous scaffolds for bone tissue engineering. Among of them, collagen guarantees good biological conditions, and ß-tricalcium phosphate (ß-TCP) possesses good oseteoconductivity, cellular adhesion, accelerated differentiation and mechanical property. In this study, rat bone marrow mesenchymal stem cells (rMSC) were cultured in ß-TCP/collagen composite scaffolds up to 28 days in order to assess the time-dependent behavior of the extracellular matrix formation and the mechanical performance of the scaffold-cell system. The cell number and ALP activity were evaluated using a spectrophotometric plate reader. Gene expression of osteogenesis was analyzed using the real-time PCR reactions. Compression tests were also conducted periodically by using a conventional testing machine to evaluate the elastic modulus. The increasing behaviors of cell number and ALP activity in the composite scaffold were much better than in the collagen scaffold. The gene expression of osteocalcin and collagen type-I in collagen/ß-TCP scaffold was higher than that of the collagen scaffold. The compressive modulus also increased up to 28 days. These results clearly showed that the distribution of micro ß-TCP particles is very effective to increase the elastic modulus and promote cell growth.

Biomechanical evaluation of porous bioactive ceramics after implantation: micro CT-based three-dimensional finite element analysis.

Hydroxyapatite ceramics have been widely investigated for bone regeneration due to their high biocompatibility. However, few studies focus on their mechanical characteristics after implantation. In this study, the finite element (FE) method was used to evaluate the mechanical properties of a fully interconnected porous hydroxyapatite (IPHA) over time of implantation. Based on the micro-CT images obtained from the experiments dealing with IPHA implanted into rabbit femoral condyles, three-dimensional FE models of IPHA (1, 5, 12, 24, and 48 weeks after implantation) were developed. FE analysis indicated that the elastic modulus gradually increased from 1 week and reached the peak value at 24 weeks, and then it kept at high level until 48 weeks postoperatively. In addition, as a local biomechanical response, strain energy density became to distribute evenly over time after the implantation. Results confirmed that the mechanical properties of IPHA are strongly correlated to bone ingrowth. The efficiency of the proposed numerical approach was validated in combination with experimental studies, and the feasibility of applying this approach to study such implanted porous bioceramics was proved.