Early Observations About Cells Adhesion And Growth On Culture Of Human Mesenchymal Stem Cells With Human Femoral Head Acellular Bone Matrices In Vitro

Objective：To investigate the interaction between humanmesenchymal stem cells (hMSCs) and two kinds of acellular bonematrices.One of the acellular bone matrices were obtained from thefemoral heads of avascular necrosis，and the other from normal femoralheads. The purpose of this trial was to find a novel viewpoint for theMSCs transplantation in the treatment of the femoral head of avascularnecrosis. Method：hMSCs were isolated from the volunteer patients’bone marrows who needed to iliac bone grafting of themselves own bywhole-bone marrow culture method，and also were identified. P1, P2, P3cells were chosen in their good state of growth and cultured for12days,then their growth curves were plotted. The femoral heads were sectionedinto sclerites from the patients who needed hip surgery for theinterception of the femoral head through coronal center, then were madeinto acellular bone matrices.The one was marked for osteonecrosis groups,the other marked for non osteonecrosis groups, and two kinds of acellularbone matrices were both stained by HE staining andimmunohistochemical processing, to ensure there was no cells in the bonematrices, and to observe the distributions of fibronectin（FN）、laminin（LN） in the acellular bone matrices.The two kinds of acellular bone matrices were devieded into equal quantity cultured with A（2×105/mL）、B（4×105/mL）、C（6×105/mL）three groups of different concentrationof P3hMSCs, then cultured for24hours,72hours and7days after theobservation of cells adhesions.Meanwhile, two kinds of acellular bonematrices were cultured with the same concentration of hMSCs for24hours, by counting the exfoliated cells, then calculated the adhesion rates;Statistical analyses were performed with SPSS17.0（P <0.05）.Toobserve the distribution of cell adhesion about24hours cultured group，we fixed the co-cultured complex paraffins and buried them by laserbefore observeded by confocal laser scanning microscope.To observe thedistribution of cells adhesion about72hours and7days cultured groups，the co-cultured complex paraffins were stained by HE staining，and alsoobserve the differences between groups with different concentrations ofcells adhesion situations compared with non osteonecrosis groups.Results:Highly purity hMSCs were obtained after separations of whole-bonemarrow cultured mathod，and hMSCs were identified matched with theirown characterations，and the growth curve was consistent with cellproliferation index like a “S”.After HE staining, acellular bone matrixswere showed no cellular components，immunohistochemical detection ofbone matrices about FN, LN in the non osteonecrosis groups acellularbone matrices in continuous reaction, but the osteonecrosis groupsshowed interruption performances.After12hours co-culturing，hMSCscompletely attached to the bottle wall or adhered to the acellular bone matrices.24hours of cell adhesion rates were compared with the sameconcentration of hMSCs culture,the mean rates of osteonecrosis groupsand non osteonecrosis groups were （44.40±3.22）%、（67.01±3.08）%.The number of adherent cells in group C was found more higher thanthat in group A after co-cultured24hours under confocal laser scanningmicroscope, including the number of osteonecrosis groups of necrosisarea of adhesion of cells were relatively less.The distribution aboutadhesion of hMSCs in the bone matrixs was different after co-cultured72hours and7days when HE staining on the complex.Non osteonecrosisgroups of cells adhesion was uniform, but osteonecrosis groups was not,near the necrosis area of cells adhesion was poor, and even no cells, andaway from this area，the cells adhesion distributed was uniform，whichmatched with non osteonecrosis groups.And we also observed withinoculation of hMSCs concentration increasing, both the osteonecrosisand non osteonecrosis of acellular bone matrix, hMSCs in acellular bonematrix on the growth of invasive depth increased, the latter invasiongreater depth.Coclusions: The hMSCs were easily isolated viawhole-bone marrow culture method，and the hMSCs were identified inline with the phenotypic characteristics. The use of two-step hypotonicand detergents can be effective in acellular bone. The acellular bonematrices of avascular necrosis were more conducive to the growth ofhMSCs adhesions and invasions, and with increasing concentrations ofhMSCs vaccinated, the number of cells in the bone matrix were increased,and the depth too.