Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with beta-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.
Adipocytes, White/cytology/physiology ; Alkaline Phosphatase/metabolism ; Animals ; Biocompatible Materials/metabolism/*therapeutic use ; Bone Diseases/*therapy ; Bone Marrow Cells/cytology/physiology ; Calcification, Physiologic ; Calcium/metabolism ; Calcium Phosphates/metabolism/therapeutic use ; Cell Proliferation ; Dogs ; Female ; Fetal Blood/cytology/physiology ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells/cytology/*metabolism ; *Osteogenesis ; Polyesters/metabolism/therapeutic use ; Tissue Engineering/*methods ; Vascular Endothelial Growth Factor A/metabolism

This study was performed to evaluate the osteogenic effect of allogenic canine umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) mixed with beta-tricalcium phosphate (beta-TCP) in orthotopic implantation. Seven hundred milligrams of beta-TCP mixed with 1 x 10(6) UCB-MSCs diluted with 0.5 ml of saline (group CM) and mixed with the same volume of saline as control (group C) were implanted into a 1.5 cm diaphyseal defect and wrapped with PLGC membrane in the radius of Beagle dogs. Radiographs of the antebrachium were made after surgery. The implants were harvested 12 weeks after implantation and specimens were stained with H&E, toluidine blue and Villanueva-Goldner stains for histological examination and histomorphometric analysis of new bone formation. Additionally, UCB-MSCs were applied to a dog with non-union fracture. Radiographically, continuity between implant and host bone was evident at only one of six interfaces in group C by 12 weeks, but in three of six interfaces in group CM. Radiolucency was found only near the bone end in group C at 12 weeks after implantation, but in the entire graft in group CM. Histologically, bone formation was observed around beta-TCP in longitudinal sections of implant in both groups. Histomorphometric analysis revealed significantly increased new bone formation in group CM at 12 weeks after implantation (p < 0.05). When applied to the non-union fracture, fracture healing was identified by 6 weeks after injection of UCB-MSCs. The present study indicates that a mixture of UCB-MSCs and beta-TCP is a promising osteogenic material for repairing bone defects.
Animals ; Biocompatible Materials/metabolism/therapeutic use ; Bone Substitutes/*therapeutic use ; Calcium Phosphates/*therapeutic use ; Dogs ; Fetal Blood/*cytology ; Fracture Fixation/methods/veterinary ; Mesenchymal Stem Cells/*physiology ; Osteogenesis/*physiology ; Tissue Engineering/methods ; Wound Healing/physiology

This study was performed to evaluate the effect of betatricalcium phosphate and poly L-lactide-co-glycolide-coepsilon- caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/ PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/ PLGC group. However, the new cortical bone in the TCP/ PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/ PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.
Absorbable Implants/veterinary ; Animals ; Bone Regeneration/*drug effects ; Calcium Phosphates/*pharmacology ; Dogs/*surgery ; Guided Tissue Regeneration/methods/*veterinary ; Histocytochemistry/veterinary ; Humerus/*surgery ; Male ; Polyesters/*pharmacology ; Tomography, X-Ray Computed/veterinary ; Wound Healing/physiology