Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice

Aim: The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology: DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results: The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion: As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo

A Novel 3D Bioprinter Using Direct-Volumetric Drop-On-Demand Technology for Fabricating Micro-Tissues and Drug-Delivery

Drop-on-demand (DOD) 3D bioprinting technologies currently hold the greatest promise for generating functional tissues for clinical use and for drug development. However, existing DOD 3D bioprinting technologies have three main limitations: (1) droplet volume inconsistency; (2) the ability to print only bioinks with low cell concentrations and low viscosity; and (3) problems with cell viability when dispensed under high pressure. We report our success developing a novel direct-volumetric DOD (DVDOD) 3D bioprinting technology that overcomes each of these limitations. DVDOD can produce droplets of bioink from <10 nL in volume using a direct-volumetric mechanism with <± 5% volumetric percent accuracy in an accurate spatially controlled manner. DVDOD has the capability of dispensing bioinks with high concentrations of cells and/or high viscosity biomaterials in either low- or high-throughput modes. The cells are subjected to a low pressure during the bioprinting process for a very short period of time that does not negatively impact cell viability. We demonstrated the functions of the bioprinter in two distinct manners: (1) by using a high-throughput drug-delivery model; and (2) by bioprinting micro-tissues using a variety of different cell types, including functional micro-tissues of bone, cancer, and induced pluripotent stem cells. Our DVDOD technology demonstrates a promising platform for generating many types of tissues and drug-delivery models

Articular Cartilage Regeneration through Bioassembling Spherical Micro-Cartilage Building Blocks

Articular cartilage lesions are prevalent and affect one out of seven American adults and many young patients. Cartilage is not capable of regeneration on its own. Existing therapeutic approaches for articular cartilage lesions have limitations. Cartilage tissue engineering is a promising approach for regenerating articular neocartilage. Bioassembly is an emerging technology that uses microtissues or micro-precursor tissues as building blocks to construct a macro-tissue. We summarize and highlight the application of bioassembly technology in regenerating articular cartilage. We discuss the advantages of bioassembly and present two types of building blocks: multiple cellular scaffold-free spheroids and cell-laden polymer or hydrogel microspheres. We present techniques for generating building blocks and bioassembly methods, including bioprinting and non-bioprinting techniques. Using a data set of 5069 articles from the last 28 years of literature, we analyzed seven categories of related research, and the year trends are presented. The limitations and future directions of this technology are also discussed

Rapid Fabrication of Anatomically-Shaped Bone Scaffolds Using Indirect 3D Printing and Perfusion Techniques

Fused deposit modeling (FDM) 3D printing technology cannot generate scaffolds with high porosity while maintaining good integrity, anatomical-surface detail, or high surface area-to-volume ratio (S/V). Solvent casting and particulate leaching (SCPL) technique generates scaffolds with high porosity and high S/V. However, it is challenging to generate complex-shaped scaffolds; and solvent, particle and residual water removal are time consuming. Here we report techniques surmounting these problems, successfully generating a highly porous scaffold with the anatomical-shape characteristics of a human femur by polylactic acid polymer (PLA) and PLA-hydroxyapatite (HA) casting and salt leaching. The mold is water soluble and is easily removable. By perfusing with ethanol, water, and dry air sequentially, the solvent, salt, and residual water were removed 20 fold faster than utilizing conventional methods. The porosities are uniform throughout the femoral shaped scaffold generated with PLA or PLA-HA. Both scaffolds demonstrated good biocompatibility with the pre-osteoblasts (MC3T3-E1) fully attaching to the scaffold within 8 h. The cells demonstrated high viability and proliferation throughout the entire time course. The HA-incorporated scaffolds demonstrated significantly higher compressive strength, modulus and osteoinductivity as evidenced by higher levels of alkaline-phosphatase activity and calcium deposition. When 3D printing a 3D model at 95% porosity or above, our technology preserves integrity and surface detail when compared with FDM-generated scaffolds. Our technology can also generate scaffolds with a 31 fold larger S/V than FDM. We have developed a technology that is a versatile tool in creating personalized, patient-specific bone graft scaffolds efficiently with high porosity, good scaffold integrity, high anatomical-shaped surface detail and large S/V

Bone marrow Derived Pluripotent Cells are Pericytes which Contribute to Vascularization

Pericytes are essential to vascularization, but the purification and characterization of pericytes remain unclear. Smooth muscle actin alpha (α-SMA) is one maker of pericytes. The aim of this study is to purify the α-SMA positive cells from bone marrow and study the characteristics of these cells and the interaction between α-SMA positive cells and endothelial cells. The bone marrow stromal cells were harvested from α-SMA-GFP transgenic mice, and the α-SMA-GFP positive cells were sorted by FACS. The proliferative characteristics and multilineage differentiation ability of the α-SMA-GFP positive cells were tested. A 3-D culture model was then applied to test their vascularization by loading α-SMA-GFP positive cells and endothelial cells on collagen-fibronectin gel. Results demonstrated that bone marrow stromal cells are mostly α-SMA-GFP positive cells which are pluripotent, and these cells expressed α-SMA during differentiation. The α-SMA-GFP positive cells could stimulate the endothelial cells to form tube-like structures and subsequently robust vascular networks in 3-D culture. In conclusion, the bone marrow derived pluripotent cells are pericytes and can contribute to vascularization

DAPT Enhances the Apoptosis of Human Tongue Carcinoma Cells

AIM: To investigate the effect of DAPT (γ-secretase inhibitor) on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma. METHODOLOGY: Human tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence (IF) were employed to determine the intracellular expression levels. RESULTS: DAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G(0)–G(1) cell cycle arrest and apoptosis. The mRNA levels of Hairy/Enhancer of Split-1 (Hes-1), a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells. CONCLUSION: DAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch-1 and Caspase-3