We assessed the efficacy of frozen-thawed gelatin-induced osteogenic cell sheet (FT-GCS) compared to that of fresh gelatin-induced osteogenic cell sheet (F-GCS) with adipose-derived mesenchymal stromal cells (Ad-MSCs) used as the control. The bone differentiation capacity of GCS has already been studied. On that basis, the experiment was conducted to determine ease of use of GCS in the clinic. In vitro evaluation of F-GCS showed 3–4 layers with an abundant extracellular matrix (ECM) formation; however, cryopreservation resulted in a reduction of FT-GCS layers to 2–3 layers. Cellular viabilities of F-GCS and FT-GCS did not vary significantly. Moreover, there was no significant difference in mRNA expressions of Runx2, β-catenin, OPN, and BMP-7 between F-GCS and FT-GCS. In an in vivo experiment, both legs of six dogs with transverse radial fractures were randomly assigned to one of three groups: F-GCS, FT-GCS, or control. Fracture sites were wrapped with the respective cell sheets and fixed with 2.7 mm locking plates and six screws. At 8 weeks after the operations, bone samples were collected and subjected to micro computed tomography and histopathological examination. External volumes of callus as a portion of the total bone volume in control, F-GCS, and FT-GCS groups were 49.6%, 45.3%, and 41.9%, respectively. The histopathological assessment showed that both F-GCS and FT-GCS groups exhibited significantly (p < 0.05) well-organized, mature bone with peripheral cartilage at the fracture site compared to that of the control group. Based on our results, we infer that the cryopreservation process did not significantly affect the osteogenic ability of gelatin-induced cell sheets.
Animals ; Bone Morphogenetic Protein 7 ; Bony Callus ; Cartilage ; Cryopreservation ; Dogs ; Extracellular Matrix ; Fracture Healing ; In Vitro Techniques ; Leg ; Mesenchymal Stromal Cells ; RNA, Messenger

Cell sheets technology is being available for fracture healing. This study was performed to clarify bone healing mechanism of undifferentiated (UCS) and osteogenic (OCS) differentiated mesenchymal stromal cell (MSC) sheets in the fracture model of dogs. UCS and OCS were harvested at 10 days of culture. Transverse fractures at the radius of six beagle dogs were assigned into three groups (n = 4 in each group) i.e. UCS, OCS and control. The fractures were fixed with a 2.7 mm locking plate and six screws. Cell sheets were wrapped around the fracture site. Bones were harvested 8 weeks after operation, then scanned by micro-computed tomography (micro-CT) and analyzed histopathologically. The micro-CT revealed different aspects of bone regeneration among the groups. The percentages of external callus volume out of total bone volume in control, UCS, and OCS groups were 42.1, 13.0 and 4.9% (p < 0.05) respectively. However, the percentages of limbs having connectivity of gaps were 25, 12.5 and 75% respectively. In histopathological assessments, OCS group showed well organized and mature woven bone with peripheral cartilage at the fracture site, whereas control group showed cartilage formation without bone maturation or ossification at the fracture site. Meanwhile, fracture site was only filled with fibrous connective tissue without endochondral ossification and bone formation in UCS group. It was suggested that the MSC sheets reduced the quantity of external callus, and OCS induced the primary bone healing.
Animals ; Bone Regeneration ; Bony Callus ; Cartilage ; Connective Tissue ; Dogs ; Extremities ; Fracture Healing ; Mesenchymal Stromal Cells ; Osteogenesis ; Radius