Freeze-dry processing of three-dimensional cell constructs for bone graft materials.

Freeze-dry processing improves the operability and stability of cell-based biomaterials and facilitates sterilization for clinical application. However, there is no established freeze-drying protocol for engineered tissues. Recently, we reported that biomimetic bone tissues can be fabricated using scaffold-free three-dimensional (3D) cell constructs with potential applications as bone graft materials. The purpose of this study was to assess the influence of freeze drying on the morphology and components of 3D cell constructs. Cell constructs freeze dried in phosphate buffered saline (PBS) maintained organic and inorganic components; whereas sodium citrate buffer (SCB)-treated constructs had significantly lower amounts of calcium and bone-related proteins. Alkaline phosphatase (ALP) activity in cell constructs was maintained by freeze drying in 10% sucrose-containing PBS, whereas cell constructs treated with PBS without sucrose or with sucrose-containing SCB showed significant reductions of ALP activity. In this study, we found that sucrose-containing phosphate buffer was suitable for freeze drying to maintain minerals and protein functions within 3D cell constructs, whereas citrate buffer was inappropriate. The insights gained by this study may facilitate the development of novel cell-based biomaterials fabricated by tissue engineering approaches and bone graft biomaterials.

Development of a novel dental resin cement incorporating FGF-2-loaded polymer particles with the ability to promote tissue regeneration.

OBJECTIVE: Aiming to achieve bioactive dental resins that promote healing of surrounding tissues, we developed novel poly(2-hydroxyethyl methacrylate/trimethylolpropane trimethacrylate) (polyHEMA/TMPT) particles. These particles have been reported to be useful as a non-biodegradable carrier for fibroblast growth factor-2 (FGF-2) in vitro. The aim of this study was to evaluate the ability of an adhesive resin incorporating FGF-2-loaded polymer particles to promote tissue regeneration in vitro and in vivo. METHODS: Experimental adhesive resins were prepared by incorporating FGF-2-loaded polyHEMA/TMPT particles into a 4-META/MMA-based adhesive resin, and the release profiles of FGF-2 were evaluated. The proliferation of osteoblast-like cells in the eluate from cured experimental resin was assessed. When the experimental resin was implanted into rat calvaria defects, bone regeneration was evaluated by microcomputed tomography and histological observations. RESULTS: Sustained release of FGF-2 from the experimental resin was observed for 14 days. Eluate from the cured experimental resin significantly promoted the proliferation of osteoblast-like cells. Significantly greater bone regeneration was observed using the experimental resin compared with the control resin without FGF-2. SIGNIFICANCE: 4-META/MMA-based adhesive resin incorporating FGF-2-loaded polymer particles is useful to promote tissue regeneration, suggesting that its application would be beneficial for root-end filling or the repair of fractured roots in cases with severely damaged periodontal tissue.

Development of layered PLGA membranes for periodontal tissue regeneration.

OBJECTIVE: Various commercial products are available for guided tissue regeneration (GTR) therapy; however, they do not combine biosafety with the ability to control cell function. The purpose of this study was to evaluate the physicochemical and biological characteristics of the novel bilayer biodegradable poly(lactic-co-glycolic acid) (PLGA) membrane, and to assess whether the bilayer PLGA membrane could be used for periodontal tissue regeneration. METHODS: Bilayer biodegradable membrane was fabricated thorough a two-step freezing and lyophilization process using PLGA solution. The characteristics of bilayer membranes were evaluated with respect to surface morphology, stability, mechanical strength, and operability for clinical use. Cell proliferation and osteogenic differentiation were investigated on the each surface of bilayer membrane. Then, these membranes were implanted to the rat calvaria bone defect models and evaluated their capability for tissue regeneration. RESULTS: Biodegradable membranes composed of the solid and porous layer were successfully prepared and the surface morphologies analyzed. Physicochemical analyses revealed that the membranes possessed enough stability and mechanical properties for clinical use. It was also confirmed that the solid layer inhibited cell proliferation and subsequent connective tissue invasion, while the inner layer promoted proliferation and osteogenic differentiation, thus resulting in bone regeneration in vivo. SIGNIFICANCE: The layering technology used to fabricate the bilayer polymer membrane could be applied in the developing of other novel biomaterials. The present study demonstrates that the bilayer biodegradable polymer membranes facilitate tissue regeneration in vivo, and therefore represent a prospective biomaterial for GTR therapy.

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells.

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.