Effects Of Different DBM Morphology On The Adhesion And Proliferation Of Osteoblasts

ObjectiveDemineralized bone matrix?DBM?is a kind of natural biological material which is based on collagen and has the properties of osteoconduction and osteoinduction.Currently the osteogenesis effectiveness remains controversial,the purpose of this research is to evaluate the biocompatibility of different morphology surface demineralized bone matrix?SDBM?such as ball,granule and fibre,by observing the adhesive,proliferative and differential behaviors of osteoblast in vitro.Providing theoretic foundations for further explore the influence of different morphology of SDBM on osteogenic properties in vivo.MethodsThe different morphology of three groups of DBM materials was observed by general observation and scanning electron microscopy?SEM?.Co-cultured three groups of DBM with MC3T3-E1 cells and using fluorescence microscope observe cells dead/live staining.Meanwhile,the growth and shape of MC3T3-E1 on DBM were observed under SEM at the 1st and 7th days following seeding.In addition,the adhesion rates of cells on different surface morphology at the 24h after seeding compared by cell count.The growth and proliferation ability of cells co-cultured with each group DBM products was tracked by CCK-8 examination at 1st,3rd,5th,7th,9th days after plating and the growth and proliferation curves was drawn.ResultsThe surface morphology and electron microscopy showed that the surface of DBM ball was smooth with sporadic distribution of pores.The DBM fiber group has a flat surface with loose fibrous structure and a dense network structure with many small pores.DBM granule has irregular uneven structure and covered with different protruding micron-scale particles.The results of dead/living cells staining showed that all of the three groups of DBM had good cytocompatibility.The results of SEM after 24h of co-culture showed that cells in shuttle shape attached and spreaded well on DBM fiber surface;Cells in granule group closely jointed and spread out more open compared with ball and fiber,cells were in thinner fibroid shape and the pseudopodia was tenuous and communicated with each other;Cells on the DBM ball had a roundness shape,loose contact with surface.There were also more cells in the granule group and fiber group compared with ball group,which fused with each other and secreted a large amount of extracellular matrix after 7th days of co-culture.Cell count showed that the highest adhesion rate was 33.1±6.5%in the fiber group,followed by bone granule group?32.1%±6.9%?and ball group?24.7%±6.9%?,but no statistically signifiant?P=0.324?.The results of CCK-8 showed that OD values of both granule group and fiber group were higher than ball group at any point in time?P>0.05?.Compared the among different groups in different time periods,we found that the OD_3⁵d?OD_5⁷d and OD_7⁹d increase of granule group was greater than fiber group and ball group?P<0.05?.And the OD value increase of ball group was the lowest.Conclusion.The adhesion,growth and proliferation of MC3T3-E1 osteoblasts on the surface of granule and fiber group were significantly better than that of ball group,which indicated that different morphology of SDBM have an important influence on the biocompatibility of cells to some extent.As a SDBM which suitable for large bone defects,weight-bearing bone defects and bone graft fusion for joints and spine,DBM granules and DBM fibers are more preferable choices.In addition,the design of DBM product should not only consider optimizing its bone conductivity and bone inductivity,but also should combine with physical factors such as morphology,structure and geometric characteristics to guide and promote cell growth,proliferation and differentiation towards the right direction,so that DBM can fully exert its ability to stimulate and guide bone tissue regeneration.