Three-dimensional Formation of Bone Tissue of Osteoblast Cells Which Carried Out Differentiation Induction from the Human Adipose Tissue Stem Cells
As a material used for the conventional bone transplantation, there are (1) autologous bone, (2) bone of others, and (3) artificial bones.
However, the autologous bone needs to take a bone from other parts, and the burden on the body becomes large. Moreover, when using others' bone, there is a problem of infection, immunity, and bioethics, and when using an artificial bone, the problem that replacing autologous bone takes time is raised.
In order to regenerate a three-dimensional bone tissue in vitro, we used aterocollagen beads and induced osteoblasts which were differentiated from the adipose tissue stem cells.
Materials and Methods
Subcutaneous adipose tissue from the abdomen were collected by suction, were taken by excision from the buccal fat pad and from the buttocks of the femoral neck fracture patients. The fragments of adipose tissue were dissociated with collagenase/dispase solution at 37℃ for 30 min. DMEM/F12 supplemented with 15% FBS was used as a growth medium (GM). After a cell reaches sub-confluent, the stem cells were isolated according to the Freidenstein’s method.
The induction differentiation (ID) of the adipose derived stem cells (ADSCs) to the osteoblast, we used ID-medium containing 0.1mM ascorbic acid, 10mM beta-glycerophosphate, and 10nM dexamethasone in GM. The area of osteoblasts was isolated by colonial cloning method.
The CD31 conjugated magnetic beads were used for isolation of endothelial cells from SVF.
The atelocollagen beads (ACBs) and induced osteoblasts, or ACBs and endothelial cells were cultured individually on the shaker for overnight. Two kinds of cell conjugated beads were mixed in a mold and cultured in differentiation-culture medium containing 1µg/ml calcitonin, 0.5µg/ml BMP2 (BMP4), 1µg/ml IGF-1 and 0.5 µg/ml of VEGF and cultured for 4 weeks.
The osteoblast-beads were transplanted into subcutaneous of mice and the tissue was observed after 3 weeks from the injection.
Methods of data analysis
The degree of differentiation of a stem cell is judged in the gene analysis by RT-PCR and an electron microscope.
The induced osteoblast and regenerated bone was examined by an optical microscope, µCT and histologically observations. Moreover, immunohistochemical staining for endothelial cell of blood vessels, osteoblasts were performed using a specific antibodies.
Results
Adipose tissue stem cells (ADSCs) were expressed Nanog, Oct3/4, and Sox2. Also, cell size was small, and cell-organelles were poor. The induced differentiated osteoblasts were large and well developed cell-organelles. These induced cells were immunostained with anti-osteonectin, osteoponchin and osteocalcin. The regenerated bone was made of small bone-fragments and wrapped in the periosteum. The intramembranous ossifications were observed. The Haversian system like structure was also observed. Inside of canals, anti-vWF positive cells were made blood vessel like structure. When osteoblast-beads were transplanted into subcutaneously, the regenerated bone was observed.
Conclusion
In order to make a bone tissue, first, a tissue stem cell is isolated from human adipose tissue, and second, this stem cell is induced to the osteoblast in vitro. Third, for regeneration of tree-dimensional bone, we use a cell-beads method. In order to make a net-works of blood vessels in the bone tissue, endothelial cell-beads are mixed in the osteoblast-beads, and put them into the mold. The Haversian system like structure was observed in the regenerative bone tissue. If a patient's own serum is used by our method, bone transplantation of custom-made is possible.
References
Guilak F, Lott KE, Awad HA, Cao Q, Hicok KC, Fermor B, Gimble JM. Clonal analysis of the differentiation potential of human adipose-derived adult stem cells. J Cell Physiol. 2006; 206(1):229-37.
Matsunaga YT, Morimoto Y, Takeuchi S. Molding cell beads for rapid construction of macroscopic 3D tissue architecture. Adv Mater. 2011; 23(12):H90-4.