Isolation, Culture And Identification Of Canine Marrow Blood EPCs And CD133 Expression Research Of EPCS In A NTDO

Objective Endothelial progenitor cells were isolated from the bone marrow of healthy puppies, then cells were identified, cultured and used as experimental materials for the experimental groups. Non-vascularized transport distraction osteogenesis animal models of dog mandible were constructed. By the expression of endothelial progenitor cells in distraction area, a process of the dog mandible non-vascularized transport distracted into distraction osteogenesis transport was preliminarily studied, as well as the blood supply reconstruction process of non-vascularized transport distraction osteogenesis.Methods Healthy 1-2 month old puppies were chosen, and marrow was extracted via tibia bone. The mononuclear cells were isolated with density gradient centrifugation using canine lymphocyte separation medium. Special inducing culture medium was used to obtain mononuclear cells and endothelial progenitor cells, and amplification culture was conducted. Inverted microscope was used to observe cells in the culture daily. Flow cytometry was used to identify the surface markers (CD34, CD 133) of target cells. Laser scanning confocal microscope was used to observe the uptake of DIL-AC-LDL combining with FITC-Lectin-UEA-1 test. MTT assay was used to observe cell proliferation activity. 12 healthy adult crossbreed dogs were randomly divided into six groups as follows:stretching group 1, fixed a week after distraction (A1), two weeks (B1), four weeks (C1) and injury group 2, after one week (A2), two weeks (B2), four weeks (C2), n=2. In the surgery, a 20.0 mm × 20.0 mm of mandibular defect was constructed at the lower edge of the right side of the dog mandible, and a 10.0 mm × 10.0 mm of non-vascularized transport was constructed in the distal end of the defects area. According to distraction group and injury group, distraction animals were installed within an internal distractor device. After 5 days,1.0 mm/day/time rate of nearly continuous distraction was started to repair the defect. In injury group (group 2), the non-vascularized animal transport was fixed directly in the defect near end with a titanium plate. After the distraction and surgery, according to the planned days, the animals were sacrificed and prepared into samples for gross observation and CD133 immunohistochemistry.ResultsIsolation, identification and amplification culture of canine bone marrow-derived endothelial progenitor cells:new isolated mononuclear cell was round, ununiformed large and small and floating in the medium. After 24 hours, part of cells began to grow adherently with fusiform shape, triangle and spindle shape. After 3 to 7 days, cell growth was quick and cell colonies were appear. Intermediate cells were more round than cells in the edge. Cells in the edge showed the sprouting like stretching, which was similar with blood island-like structure. After 8 to 12 days, the cells gradually grew fully in the flasks, showed a typical cobblestone appearance and cells were paving stone like. After 14 days, cells were arranged tightly, and with capillary tube-like structure. Flow cytometry results showed that cells were with CD34 and CD 133 positive expression. Cell growth curve was "S" shape. Cells were capable of uptaking DIL-AC-LDL combining with FITC-Lectin-UEA-1. EPCs in the expression study of non-vascularized transport distracted into osteogenesis distraction:all experimental animals completed distraction osteogenesis process without wound infection, and the animals were survived to the planned sampling time. Internal distractor device fixed to the mandibular was without loosening and without damage. By CD133 immunohistochemistry detecting, the positive rate of A was greater than B, while the positive rate showed as follow:one week group> two weeks group>four weeks group.Conclusion1. A methodology of canine bone marrow-derived endothelial progenitor c ells isolation, culture and identification was preliminarily established.2. The EPCs numbers in non-vascularized transport distraction osteogenesi s area was less than the EPCs numbers in the damage area of the injury model in the corresponding period.