Canine Bone Marrow-derived Mesenchymal Stem Research Articles

Compensatory Cellular Reactions to Nonsteroidal Anti-Inflammatory Drugs on Osteogenic Differentiation in Canine Bone Marrow-Derived Mesenchymal Stem Cells

ABSTRACTThe suppressive effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the bone healing process have remained controversial, since no clinical data have clearly shown the relationship between NSAIDs and bone healing. The aim of this study was to assess the compensatory response of canine bone marrow-derived mesenchymal stem cells (BMSCs) to several classes of NSAIDs, including carprofen, meloxicam, indomethacin and robenacoxib, on osteogenic differentiation. Each of the NSAIDs (10 µM) was administered during 20 days of the osteogenic process with human recombinant IL-1β (1 ng/ml) as an inflammatory stimulator. Gene expression of osteoblast differentiation markers (alkaline phosphatase and osteocalcin), receptors of PGE2 (EP2 and EP4) and enzymes for prostaglandin (PG) E2 synthesis (COX-1, COX-2, cPGES and mPGES-1) was measured by using quantitative reverse transcription-polymerase chain reaction. Protein production levels of alkaline phosphatase, osteocalcin and PGE2 were quantified using an alkaline phosphatase activity assay, osteocalcin immunoassay and PGE2 immunoassay, respectively. Histologic analysis was performed using alkaline phosphatase staining, von Kossa staining and alizarin red staining. Alkaline phosphatase and calcium deposition were suppressed by all NSAIDs. However, osteocalcin production showed no significant suppression by NSAIDs. Gene expression levels of PGE2-related receptors and enzymes were upregulated during continuous treatment with NSAIDs, while certain channels for PGE2 synthesis were utilized differently depending on the kind of NSAIDs. These data suggest that canine BMSCs have a compensatory mechanism to restore PGE2 synthesis, which would be an intrinsic regulator to maintain differentiation of osteoblasts under NSAID treatment.

Evaluation of Canine Bone Marrow-derived Mesenchymal Stem Cells After Long-term Cryopreservation

Autologous bone marrow-derived mesenchymal stem cell (BMSCs)-based therapies show great potential in regenerative medicine. However, long-term storage and preservation of BMSCs for clinical use is still a great clinical challenge. The present study aimed to analyze the effect of long-term cryopreservation on the regenerative ability of BMSCs. After cryopreservation of BMSCs from beagle dogs for three years, cell viability, and quantitative analysis of alkaline phosphatase (ALP) activity, surface adherence, and mineralized nodule formation were analyzed. BMSCs in cell-scaffold complex were then implanted into nude mice. There was no significant difference in cell viability and ALP activity between osteogenic differentiation and non-osteogenic differentiation of BMSCs, and BMSCs in cell-scaffold complex retained osteogenic differentiation ability in vivo. These results indicate that long-term cryopreserved BMSCs maintain their have capacity to contribute to regeneration.

Isolation, characterization and transduction of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs)

BACKGROUND: Stem cell therapy in small animal medicineis still in its infancy and few in vitro and in vivo research projectsregarding animal Mesenchymal Stem Cells (MSCs) have beencarried out. On the other hand, Cell tracking is the first step of thecell-based therapies and is essential to recognize cell fate posttransplantation. OBJECTIVES: The aim of this study was toisolate, characterize, and transduce cBM-MSCs. METHODS:Canine Bone Marrow-derived Mesenchymal Stem Cells (cBMMSCs)were isolated from bone marrow of dogs andcharacterized based on morphology, differentiation capacities,and surface marker expressions. For the first time, we labeledcBM-MSCs by GFP-encoding lentiviral vector to track them.RESULTS: cBM-MSCs were successfully isolated and proliferated.Morphologically, these cells were similar to otherMSCs from other sources and species and were able todifferentiate into osteocytes and adipocytes. cBM-MSCsexpressed surface marker CD44 but were not able to expressCD34. Approximately, 70% of cells efficaciously expressedGFPafter labeling; CONCLUSIONS:We found that GFP labelingis an easy and effective technique to track transplanted cBMMSCs.Our results also provide fundamental information aboutcanine BM-MSCs in order to use in veterinary medicine.

The Transient effects of COX-2 inhibitor on osteogenic differentiation in canine bone marrow-derived mesenchymal stem cells

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S107 differences were found between limbs for the external sagittal and frontal plane moments at the hip joint, subjects demonstrated greater external adduction moment at the knee of the SX side compared to the NSX side (MD: 0.12 0.11 Nm/Kg, p1⁄4.013, d1⁄41.09). Conclusions: The Hip Harris Score, pain level, and abductor strength suggest that the SX hip is more impaired than the NSX limb. Subjects also ambulate with a compensatory lateral trunk lean toward the affected limb, but continue to demonstrate greater adduction angles on the SX limb during single limb stance. These movement abnormalities may represent the primary movement impairments and may be related to the unilateral hip abductor weakness observed in this sample. Unilateral impairments may also contribute to the significantly larger knee adduction moment on the NSX limb. Figure 1. Average trunk lean angle (A) and hip frontal plane angle (B) during the stance phase of gait for the surgical limb (solid black line) and non-surgical limb (dashed red line). Positive angles represent lean toward the stance leg (A) and adduction (B). Black-striped area represents the standard deviation for the surgical limb. Red-squared area represents the standard deviation for the non-surgical limb. 185 THE TRANSIENT EFFECTS OF COX-2 INHIBITOR ON OSTEOGENIC DIFFERENTIATION IN CANINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS N. Oh, T. Sunaga, S. Kim, K. Hosoya, M. Okumura. Hokkaido Univ.,

A Comparison of Neurosphere Differentiation Potential of Canine Bone Marrow-Derived Mesenchymal Stem Cells and Adipose-Derived Mesenchymal Stem Cells

Stem cell transplantation is one of the most promising yet enigmatic treatments for spinal cord injury (SCI), a common problem in dogs. As pre-differentiated mesenchymal stem cells (MSCs) can be expanded and differentiated into neurospheres in vitro, before being transplanted back, they may prove to be more beneficial for treating SCI. Therefore, we compared the endogenous differentiation potential, including the neuronal cell differentiation, of neurospheres from canine bone marrow MSCs (cBMMSCs) with that of the adipose tissue-derived MSCs (cADMSCs). Nestin-positive neurospheres were generated from MSCs derived from the bone marrow and adipose tissue. Neuronal cells were differentiated from the neurospheres derived from both these tissues. Gene expression analysis revealed that Nestin, βIII-tubulin, NCAM, OCT4 and SOX2 were expressed in MSCs and the corresponding neurospheres. Notably, cBMMSC-derived neuronal cells expressed higher levels of βIII-tubulin. The mRNA expressions of NANOG, Nestin, OCT4 and SOX2 were upregulated in neurospheres derived from both. Immunofluorescence analysis detected the expression of neuronal markers, namely, βIII-tubulin, GFAP, S100, NF200 and MAP2, in differentiated neuron-like cells. Our findings highlight that both cBMMSCs and cADMSCs could be differentiated into neurospheres and neuron-like cells, and therefore, these cells are suitable candidates for cell transplantation. Further, cADMSCs form a more suitable cell source, as larger number of cells could be harvested from cADMSC-derived neurospheres. Future studies employing in vivo transplantation models to investigate the effectiveness of MSCs for treating SCI are warranted.

Osteogenic Differentiation and Ectopic Bone Formation of Canine Bone Marrow-Derived Mesenchymal Stem Cells in Injectable Thermo-Responsive Polymer Hydrogel

This study describes an injectable, thermo-responsive hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) copolymer for bone tissue engineering. The wettability, temperature-dependent change of water content, and volume of HA-CPN hydrogel were measured, together with its biocompatibility in vitro and in vivo. The dried hydrogel morphology shows a three-dimensional, porous structure with interconnected pores. Canine bone marrow-derived mesenchymal stem cells (cBMSCs) were encapsulated in HA-CPN hydrogel and osteoinduction was assessed by comparing samples with different osteogenic differentiation induction times but with the same total cell culture time. Cell proliferation and time-dependent osteogenic differentiation, evident from secretion of extracellular matrix and formation of mineral deposits, were observed. The cells showed better proliferation in HA-CPN hydrogel than on tissue culture polystyrene after osteo-induced for 21 days and higher alkaline phosphatase activity regardless of osteo-induction times. Mineralization extent of cBMSCs in HA-CPN followed by Alizarin red stains showed positive stained nodules after osteo-induced longer than 7 days. The cells/hydrogel construct also showed increased mechanical strength and elasticity after osteogenic differentiation, and the increase could be correlated with osteo-induction time. In vivo studies confirmed the biocompatibility and bioresorption of the HA-CPN hydrogel and ectopic bone formation when the hydrogel was used as a cell carrier for osteo-induced cBMSCs and implanted in nude mice subcutaneously. Taken together, the results indicate the feasibility and efficacy of HA-CPN hydrogel as an injectable bone tissue engineering scaffold with cBMSCs.