Development of robotic dispensed bioactive scaffolds and human adipose-derived stem cell culturing for bone tissue engineering.

Bioactive and degradable scaffolds made from bioactive glass-polycaprolactone with a mineralized surface and a well-defined three-dimensional (3D) pore configuration were produced using a robotic dispensing technique. Human adipose-derived stem cells (hASCs) were cultured on the 3D scaffolds, and the osteogenic development of cells within the scaffolds was addressed under a dynamic flow perfusion system for bone tissue engineering. The bioactive glass component introduced within the composite assisted in the surface mineralization of the 3D scaffolds. The hASCs initially adhered well and grew actively over the mineralized surface, and migrated deep into the channels of the 3D scaffold. In particular, dynamic perfusion culturing helped the cells to proliferate better on the 3D structure compared to that under static culturing condition. After 4 weeks of culturing by dynamic perfusion, the cells not only covered the scaffold surface completely but also filled the pore channels bridging the stems. The osteogenic differentiation of the hASCs with the input of osteogenic factors was stimulated significantly by the dynamic perfusion flow, as determined by alkaline phosphate expression. Overall, the culturing of hASCs upon the currently developed 3D scaffold in conjunction with the dynamic perfusion method may be useful for tissue engineering of bone.

FGF2-adsorbed macroporous hydroxyapatite bone granules stimulate in vitro osteoblastic gene expression and differentiation.

Hydroxyapatite bone granules with a macroporous structure were produced and then adsorbed with basic fibroblast growth factor (FGF2). The in vitro scaffolding role of the granules in cell population and osteogenic differentiation was investigated. The FGF2-adsorbed porous granules allowed the MC3T3-E1 cells to adhere well and then proliferate actively. While the cell growth level on the FGF2-treated granules was observed to be similar to that on the untreated granules, the expression of genes associated with bone, including collagen type I, alkaline phosphatase, and osteocalcin was significantly upregulated by the FGF2 treatment, particularly at the early stage. Moreover, the production of alkaline phosphatase with prolonged culturing was greatly enhanced on the FGF2-adsorbed granules. Taken together, the FGF2 treatment of the hydroxyapatite granules was effective in the osteogenic development and the FGF2-adsorbed bone granules may be useful in bone regeneration area.

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells.

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(epsilon-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA-PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA-PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA-PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA-PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering.

Nanofibrous membrane of collagen-polycaprolactone for cell growth and tissue regeneration.

Nanofibrous substrates of synthetic polymers including polycaprolactone (PCL) have shown considerable potential in tissue regeneration. This paper reports the use of PCL/collagen nanofibers to improve the in vitro osteoblastic responses for the applications in bone regeneration area. Collagen and PCL were dissolved in a co-solvent, and the resulting solution was electrospun into a nanofibrous web. Nonwoven fibrous matrices were successfully produced at various compositional ratios (PCL/collagen = 1/3, 1 and 3 by weight). Although the PCL nanofiber was hydrophobic, the presence of collagen significantly improved the water affinity, such as the water contact angle and water uptake capacity. Tensile mechanical tests showed that the collagen-PCL nanofiber had a significantly higher extension rate (approximately 2.8-fold) than the PCL while maintaining the maximum tensile load in a similar range. The osteoblastic cells cultured on the collagen-PCL nanofibrous substrate showed better initial adhesion and a higher level of growth than those cultured on the PCL nanofiber. Furthermore, real-time RT-PCR revealed the expression of a series of bone-associated genes, including osteopontin, collagen type I and alkaline phosphatase. The expression of these genes was significantly higher on the collagen-PCL nanofiber than on the PCL nanofiber. When subcutaneously implanted in mouse the collagen-PCL membrane facilitated tissue cells to well penetrate into the nanofibrous structure at day 7, whilst no such cell penetration was noticed in the pure PCL nanofiber. Overall, the presence of collagen within the PCL nanofiber improves the water affinity, tensile extension rate, and the tissue cell responses, such as initial adhesion, growth, penetration and the expression of bone-associated genes. Therefore, the collagen-PCL nanofibrous membrane may have potential applications in the cell growth and bone tissue regeneration.