Paracrine effect of canine allogenic umbilical cord blood-derived mesenchymal stromal cells mixed with beta-tricalcium phosphate on bone regeneration in ectopic implantations.

BACKGROUND AIMS: The aim of this study was to evaluate the paracrine effects of canine umbilical cord blood (cUCB) mesenchymal stromal cells (MSC) mixed with beta-tricalcium phosphate (beta-TCP) on bone regeneration in ectopic implantation. METHODS: beta-TCP mixed with cUCB MSC (UCB-MSC group), cell lysates (cell lysate group) or a control (control group) were respectively implanted in a subcutaneous pouches in the back of beagle dogs . The implants were harvested 1, 4, 7, 14, 28, 56, 84 days after implantation. Histological findings and stain analyzes of tartrate-resistant acid phosphatase (TRACP) and assays of alkaline phosphatase (ALP) and TRACP were evaluated. The mRNA expression levels of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were analyzed using semi-quantitative reverse transcription - polymerase chain reactions (RT-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to confirm the protein expression levels of IL-6, COX-2, VEGF and TGF-beta. RESULTS: TRACP-positive cells were observed in all groups 7 days after implantation. ALP and TRACP activities in the UCB-MSC group 84 days after implantation were significantly higher than those of the control (P>0.05). Histologic findings after 84 days showed that the osteoid matrix area in the UCB-MSC group was significantly larger than that of the control (P<0.05). The mRNAs levels of IL-1, IL-6 and VEGF in UCB-MSC and cell lysate groups on day 1 were up-regulated compared with the control. The protein levels of IL-6 and VEGF in the UCB-MSC group at day 1 were significantly higher than that of the other groups (P<0.05). CONCLUSIONS: It is suggested that a significant release of cytokines by cUCB MSC, 1 day following implantation, could enhance bone regeneration.

Enterocutaneous fistula as a result of chronic bite wound repair in a dog.

A 6-year-old castrated male Maltese weighing 4.8 kg was presented with a non-healing wound exhibiting purulent discharge after surgery on scar tissue of a chronic twelve-month-old bite wound on the left caudal abdominal region. The dog had previously undergone four surgeries and had been on continuous antibiotic therapy for eight months. Following radiographic and ultrasonographic examinations, the problem was diagnosed as an enterocutaneous fistula of a herniated bowel loop under the skin. Surgical resection of the fistula involving the bowel loop resolved all symptoms.

Implantation of canine umbilical cord blood-derived mesenchymal stem cells mixed with beta-tricalcium phosphate enhances osteogenesis in bone defect model dogs.

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.

Transplantation of canine umbilical cord blood-derived mesenchymal stem cells in experimentally induced spinal cord injured dogs.

This study was to determine the effects of allogenic umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) and recombinant methionyl human granulocyte colony-stimulating factor (rmhGCSF) on a canine spinal cord injury model after balloon compression at the first lumbar vertebra. Twenty-five adult mongrel dogs were assigned to five groups according to treatment after a spinal cord injury: no treatment (CN); saline treatment (CP); rmhGCSF treatment (G); UCB-MSCs treatment (UCB-MSC); co-treatment (UCBG). The UCBMSCs isolated from cord blood of canine fetuses were prepared as 10(6) cells/150 microl saline. The UCB-MSCs were directly injected into the injured site of the spinal cord and rmhGCSF was administered subcutaneously 1 week after the induction of spinal cord injury. The Olby score, magnetic resonance imaging, somatosensory evoked potentials and histopathological examinations were used to evaluate the functional recovery after transplantation. The Olby scores of all groups were zero at the 0-week evaluation. At 2 week after the transplantation, the Olby scores in the groups with the UCB-MSC and UCBG were significantly higher than in the CN and CP groups. However, there were no significant differences between the UCB-MSC and UCBG groups, and between the CN and CP groups. These comparisons remained stable at 4 and 8 week after transplantation. There was significant improvement in the nerve conduction velocity based on the somatosensory evoked potentials. In addition, a distinct structural consistency of the nerve cell bodies was noted in the lesion of the spinal cord of the UCB-MSC and UCBG groups. These results suggest that transplantation of the UCB-MSCs resulted in recovery of nerve function in dogs with a spinal cord injury and may be considered as a therapeutic modality for spinal cord injury.

Establishment of a canine spinal cord injury model induced by epidural balloon compression.

A model that provides reproducible, submaximal yet sufficient spinal cord injury is needed to allow experiments leading to development of therapeutic techniques and prediction of clinical outcome to be conducted. This study describes an experimental model for spinal cord injury that uses three different volumes of balloon inflation and durations of compression to create a controlled gradation outcome in adult dogs. Twenty-seven mongrel dogs were used for this study. A 3-french embolectomy catheter was inserted into the epidural space through a left hemilaminectomy hole at the L(4) vertebral arch. Balloons were then inflated with 50, 100, or 150 microgl of a contrast agent at the L1 level for 6, 12, or 24 h and spinal canal occlusion (SCO) measured using computed tomography. Olby score was used to evaluate the extent of spinal cord injury and a histopathologic examination was conducted 1 week after surgery. The SCO of the 50, 100, and 150 microgl inflations was 22-46%, 51-70%, and 75-89%, respectively (p < 0.05). Olby scores were diminished significantly by a combination of the level of SCO and duration of inflation in all groups. Olby scores in the groups of 150 microgl-12 h, 150 microgl-24 h, and 100 microgl-24 h were 0.5, 0, and 1.7, respectively. Based on these results, a SCO > 50% for 24 h, and > 75% for 12 h induces paraplegia up to a week after spinal cord injury.

Identification of differentially expressed genes in gauze-exposed omentum of dogs using differential display RT-PCR.

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.

Comparison of canine umbilical cord blood-derived mesenchymal stem cell transplantation times: involvement of astrogliosis, inflammation, intracellular actin cytoskeleton pathways, and neurotrophin-3.

Canine mesenchymal stem cells (cMSCs) derived from umbilical cord blood represent a potentially useful source of stem cells for therapy. The aim of this study was to compare the effects of different transplantation times of cMSCs after spinal cord injury (SCI). A total of 21 dogs were subjected to SCI by balloon-induced compression of the first lumbar vertebrae for 12 h. Of the 21 dogs, 12 were divided into four groups of three according to the time of stem cell (1 × 10(6)) transplantation at the injury site: control no treatment, 12 h, 1 week, and 2 weeks. The remaining 9 animals were negative harvest (HA) time controls for each treatment group (n = 3). Olby and Tarlov scores were used to evaluate functional recovery of the hindlimbs. Markers for neuronal regeneration (Tuj-1, nestin, MAP2, and NF-M), astrogliosis (GALC, GFAP, and pSTAT3), signal molecules for actin cytoskeleton (RhoA, Cdc42, and Rac1), inflammation (COX-2), and neurotrophins (NT-3) were evaluated by Western blot analysis. Scores of the 1-week transplantation group showed significant improvement compared to controls. Hematoxylin and eosin (H&E) staining revealed less fibrosis at the injury site in the 1-week transplantation group compared to other groups and immunohistochemistry showed increased expression of neuronal markers. Furthermore, in both 1-week and 2-week transplantation groups, Tuj-1, nestin, MAP2, NF-M, NT-3, and GFAP increased, but pSTAT3, GALC, and COX2 decreased. RhoA decreased and Rac1 and Cdc42 increased in the 1-week transplantation group. In conclusion, transplantation of cMSCs 1 week after SCI was more effective in improving clinical signs and neuronal regeneration and reducing fibrosis formation compared to the other transplantation times evaluated. Subsequently, these data may contribute to the optimization of timing for MSC transplantation used as a therapeutic modality.

Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury.

In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p < 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p < 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells.