Oh-Kyeong Kweon

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.

The fabrication and biochemical evaluation of alumina reinforced calcium phosphate porous implants.

Alumina reinforced calcium phosphate porous implants were manufactured to improve the mechanical strength while maintaining the bioactivity of calcium phosphate ceramics. The alumina porous bodies, which provided the mechanical strength, were fabricated by a polyurethane sponge method and multiple coating techniques resulted in the porous bodies with a 90-75% porosity and a compressive strength of up to approximately 6MPa. The coating of hydroxyapatite (HAp) or tricalcium phosphate (beta-TCP) was performed by dipping the alumina porous bodies into calcium phosphate ceramic slurries and sintering the specimens. The fairly strong bonding between the HAp or TCP coating layer and the alumina substrate was obtained by repeating the coating and sintering processes. The biochemical evaluations of the porous implants were conducted by in vitro and in vivo tests. For in vitro test, the implants were immersed in Ringers solution and the release of Ca and P ions were detected and compared with those of calcium phosphate powders. For in vivo test, the porous bodies were implanted into mixed breed dogs and bone mineral density measurements and histological studies were conducted. The alumina reinforced HAp porous implants had a higher strength than the HAp porous implants and exhibited a similar bioactivity and osteoconduction property to the HAp porous implants.

Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells.

BACKGROUND AIMS Previous studies have reported that scaffold or cell-based transplantation may improve functional recovery following spinal cord injury (SCI), but these results were based on neuronal regeneration and cell replacement. In this study, we investigated whether a combination of Matrigel and neural-induced mesenchymal stem cells (NMSC) improved hindlimb function in dogs with SCI, and what mechanisms were involved. METHODS We pre-differentiated canine adipose-derived mesenchymal stem cells into NMSC. A total of 12 dogs subjected to SCI procedures were assigned to one of the following three transplantation treatment groups: phosphate-buffered saline (PBS); Matrigel; or Matrigel seeded with NMSC. Treatment occurred 1 week after SCI. Basso, Beattie and Bresnahan (B.B.B.) and Tarlov scores, histopathology, immunofluorescence staining and Western blot analysis were used to evaluate the treatment effects. RESULTS Compared with dogs administered PBS or Matrigel alone, dogs treated with Matrigel + NMSC showed significantly better functional recovery 8 weeks after transplantation. Histology and immunochemical analysis revealed that the combination of Matrigel + NMSC reduced fibrosis from secondary injury processes and improved neuronal regeneration more than the other treatments. In addition, the combination of Matrigel + NMSC decreased the expression of inflammation and/or astrogliosis markers. Increased expressions of intracellular molecules related to neuronal extension, neuronal markers and neurotrophic factors were also found in the Matrigel + NMSC group. However, the expression of nestin as a neural stem cell marker was increased with Matrigel alone. CONCLUSIONS The combination of Matrigel + NMSC produced beneficial effects in dogs with regard to functional recovery following SCI through enhancement of anti-inflammation, anti-astrogliosis, neuronal extension and neuronal regeneration effects.

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 × 106) 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.

Enhanced wound healing effect of canine adipose-derived mesenchymal stem cells with low-level laser therapy in athymic mice

BACKGROUND Adipose-derived mesenchymal stem cells (ASCs) are attractive cell source for skin tissue engineering. However, one obstacle to this approach is that the transplanted ASC population can decline rapidly in the recipient tissue. OBJECTIVE The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted canine ASCs in a skin wound animal model. METHODS LLLT, ASC transplantation (ASCs) and ASC transplantation with LLLT (ASCs+LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining. The survival, differentiation and secretion of vascular endothelial growth factor and basic fibroblast growth factor of the ASCs were evaluated by immunohistochemistry and Western blotting. RESULTS The ASCs and ASCs+LLLT groups stimulated wound closure and histological skin regeneration. The ASCs+LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the ASCs group. The ASCs contributed skin regeneration via differentiation and secretion of growth factors. In the ASCs+LLLT group, the survival of ASCs was increased by the decreased apoptosis of ASCs in the wound bed. The secretion of growth factors was stimulated in the ASCs+LLLT group compared with the ASCs group. CONCLUSION These data suggest that LLLT is an effective biostimulator of ASCs in wound healing that enhances the survival of ASCs and stimulates the secretion of growth factors in the wound bed.

Low-level laser therapy promotes the osteogenic potential of adipose-derived mesenchymal stem cells seeded on an acellular dermal matrix.

This study investigates the feasibility of using an adipose-derived mesenchymal stem cell (ASC)-seeded acellular dermal matrix (ADM) along with low-level laser therapy (LLLT) to repair bone defect in athymic nude mice. Critical-sized calvarial defects were treated either with ADM, ADM/LLLT, ADM/ASCs, or ADM/ASCs/LLLT. In micro-computed tomography scans, the ADM/ASCs and the ADM/ASCs/LLLT groups showed remarkable bone formation after 14 days. Additionally, bone regeneration in the ADM/ASCs/LLLT group was obvious at 28 days, but in the ADM/ASCs group at 56 days. Bone mineral density and bone tissue volume in the ADM/ASCs/LLLT group significantly increased after 7 days, but in the ADM/ASCs group after 14 days. Histological analysis revealed that the defects were repaired in the ADM/ASCs and the ADM/ASCs/LLLT group, while the defects in the ADM and the ADM/LLLT groups exhibited few bone islands at 28 and 56 days. The successful seeding of ASCs onto ADM was confirmed, and LLLT enhanced the proliferation and the survival of ASCs at 14 days. Our results indicate that ASC-seeded grafts promote bone regeneration, and the application of LLLT on ASC-seeded ADM results in rapid bone formation. The implantation of an ASC-seeded ADM combined with LLLT may be used effectively for bone regeneration.

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.

Comparison of Osteogenesis between Adipose-Derived Mesenchymal Stem Cells and Their Sheets on Poly-ε-Caprolactone/β-Tricalcium Phosphate Composite Scaffolds in Canine Bone Defects

Multipotent mesenchymal stem cells (MSCs) and MSC sheets have effective potentials of bone regeneration. Composite polymer/ceramic scaffolds such as poly-ε-caprolactone (PCL)/β-tricalcium phosphate (β-TCP) are widely used to repair large bone defects. The present study investigated the in vitro osteogenic potential of canine adipose-derived MSCs (Ad-MSCs) and Ad-MSC sheets. Composite PCL/β-TCP scaffolds seeded with Ad-MSCs or wrapped with osteogenic Ad-MSC sheets (OCS) were also fabricated and their osteogenic potential was assessed following transplantation into critical-sized bone defects in dogs. The alkaline phosphatase (ALP) activity of osteogenic Ad-MSCs (O-MSCs) and OCS was significantly higher than that of undifferentiated Ad-MSCs (U-MSCs). The ALP, runt-related transcription factor 2, osteopontin, and bone morphogenetic protein 7 mRNA levels were upregulated in O-MSCs and OCS as compared to U-MSCs. In a segmental bone defect, the amount of newly formed bone was greater in PCL/β-TCP/OCS and PCL/β-TCP/O-MSCs/OCS than in the other groups. The OCS exhibit strong osteogenic capacity, and OCS combined with a PCL/β-TCP composite scaffold stimulated new bone formation in a critical-sized bone defect. These results suggest that the PCL/β-TCP/OCS composite has potential clinical applications in bone regeneration and can be used as an alternative treatment modality in bone tissue engineering.

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28 days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p < 0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.

Transplantation of adipose derived mesenchymal stem cells for acute thoracolumbar disc disease with no deep pain perception in dogs.

Thirty-four dogs with no deep pain perception due to acute thoracolumbar intervertebral disc disease underwent decompression surgery within 1 week of diagnosis. All dogs underwent hemilaminectomy. Adipose derived mesenchymal stem cells (AD-MSCs) were transplanted into the injured spinal cord parenchyma for the AD-MSCs transplant dogs. Long-term outcome was evaluated at the end of the follow-up period (> 6 months). AD-MSCs combination treatment showed better recovery outcomes compared to decompression surgery alone. These results indicate that this stem cell therapy is a potential therapeutic strategy to overcome the limitations of treatment for spinal cord injury in clinical medicine.