Vascularization of a Bone Organoid Using Dental Pulp Stem Cells.

Bone organoids offer a novel path for the reconstruction and repair of bone defects. We previously fabricated scaffold-free bone organoids using cell constructs comprising only bone marrow-derived mesenchymal stem cells (BMSCs). However, the cells in the millimetre-scale constructs were likely to undergo necrosis because of difficult oxygen diffusion and nutrient delivery. Dental pulp stem cells (DPSCs) are capable of differentiating into vascular endothelial lineages and have great vasculogenic potential under endothelial induction. Therefore, we hypothesized that DPSCs can serve as a vascular source to improve the survival of the BMSCs within the bone organoid. In this study, the DPSCs had greater sprouting ability, and the proangiogenic marker expressions were significantly greater than those of BMSCs. DPSCs were incorporated into the BMSC constructs at various ratios (5%-20%), and their internal structures and vasculogenic and osteogenic characteristics were investigated after endothelial differentiation. As a result, the DPSCs are differentiated into the CD31-positive endothelial lineage in the cell constructs. The incorporation of DPSCs significantly suppressed cell necrosis and improved the viability of the cell constructs. In addition, lumen-like structures were visualized by fluorescently labelled nanoparticles in the DPSC-incorporated cell constructs. The vascularized BMSC constructs were successfully fabricated using the vasculogenic ability of the DPSCs. Next, osteogenic induction was initiated in the vascularized BMSC/DPSC constructs. Compared with only BMSCs, constructs with DPSCs had increased mineralized deposition and a hollow structure. Overall, this study demonstrated that vascularized scaffold-free bone organoids were successfully fabricated by incorporating DPSCs into BMSC constructs, and the biomimetic biomaterial is promising for bone regenerative medicine and drug development.

Fabrication of novel poly(lactic acid/caprolactone) bilayer membrane for GBR application.

OBJECTIVE: Guided bone regeneration (GBR) often involves the use of membranes as barriers for soft tissues. Commercially available membranes, however, do not possess an adequately low degradation rate, resulting in limited barrier function. The purpose of this study was to develop and assess the physicochemical and biological characteristics of a novel poly(l-lactic acid/caprolactone) (PLCL) bilayer membrane and determine its usefulness for GBR application. METHODS: The experimental bilayer membrane was prepared via a two-step freezing and lyophilization process with a PLCL solution. Next, the PLCL membrane was investigated regarding tensile strength, surface roughness, in vitro degradation and clinical operability. In addition, cell proliferation and differentiation were investigated on each layer of the experimental membrane. For all experiments, a commercially available poly(lactic-co-glycolic) acid membrane was used as a control. RESULTS: In vitro analysis of the PLCL bilayer membrane revealed suitable mechanical strength combined with high breaking strain, which contributed to membrane operability. In addition, the PLCL bilayer membrane had enhanced stability compared to the commercial control due to its slower degradation, and was capable of supporting cell growth and osteogenic differentiation. SIGNIFICANCE: The current study confirmed that the PLCL membrane possessed a high biocompatibility and slow degradation rate that contributes to prolonged barrier function and bone regeneration. Altogether, it was considered that the PLCL bilayer membrane developed in this study was applicable for GBR treatment.

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.

Fabrication of strontium-releasable inorganic cement by incorporation of bioactive glass.

OBJECTIVES: Bioactive glass (BG) is widely used as a bioactive material for various clinical applications, and effective and efficient elemental release and an increase in mechanical strength are expected with further development. The purpose of this study is to clarify the physicochemical and biological characteristics of Sr-doped BG-incorporated glass ionomer cements. METHODS: Sr-doped BGs (45SiO2-6P2O5-24.5Na2O-(24.5-x)CaO-xSrO) (wt%), where × = 0, 6, 12, were prepared, and the particle size, crystallinity, and elemental release profiles were evaluated. The Sr-doped BGs were then incorporated into a glass ionomer cement at a weight ratio of 1:4, and the physicochemical properties (compressive strength, bending strength, hardness, and elemental release profile) were investigated. Cell attachment, cell proliferation, and osteoblastic differentiation were used to evaluate the biological characteristics. RESULTS: The Sr-doped BGs were amorphous phases with a homogeneous particle size and exhibited sustained release of Ca, Si, and Sr. The BG-incorporated cements were able to release these elements while retaining the same mechanical properties as those of the pure glass ionomer cement. In addition, no cytotoxicity of osteoblasts or differences in the cell attachment or proliferation were observed for the BG-incorporated cements. In contrast, the Sr-doped BG-incorporated cements promoted the alkaline phosphatase activities of the osteoblasts without the need for any media supplements for osteoblastic differentiation. SIGNIFICANCE: Sr-releasable inorganic cements with high mechanical properties were successfully fabricated by incorporating Sr-doped BGs in glass ionomer cement. These bioactive materials are promising candidates for bone grafting materials, bone cements, and pulp capping materials.

Fabricating large-scale three-dimensional constructs with living cells by processing with syringe needles.

Three-dimensional (3D) cell constructs composed only of cells and cell-secreted extracellular matrix have been attractive biomaterials for tissue engineering technology; however, controlling construct morphology and eliminating dead cells after fabrication remain a challenge. It has been hypothesized that moderate stress could shape constructs and eliminate dead cells. The purpose of this study was to establish an easily available technology for shaping 3D cell constructs and eliminating dead cells postfabrication. To achieve these objectives, spherical cell constructs composed of L-929 fibroblasts were processed using different sized syringe needles. Our results revealed that large-scale rod-shaped cell constructs could be fabricated, and that their diameters could be controlled according to the size of the syringe needle. Additionally, cell viability assays showed that >94% of cells in the rod-shaped constructs were viable, suggesting that dead cells, which have low adhesion force, were dispersed when compressive stress was applied during passage through the needle. The technology described in this study will be promising for future tissue engineering, especially for fabricating elongated tissues such as nerves and blood vessels. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 904-909, 2019.