Human Adipose Stem Cells Nanobacteria Cellulose Membrane Osteogenesis Differentiation

Objectives:to explore the feasibility of using human adipose-derived stem cells (hASCs) and bacterial cellulose(BC) as seeding cells and scaffold in the field of bone tissue engineering.Methods:1. Ultrastructure of BC membrane was observed by scanning electron microscope2. Growth pattern and morphology was observed by invert light microscope3. The proliferation of hASCs on BC membrane was investigated by CCK-8toolkit4. Adhesion of hASCs with BC membrane was investigated by frozen sectioning/HE staining and SEM of the hASCs-BC membrane complex5. Growth pattern and morphology of hASCs in the process of osteogenic differentiation was observed by light microscope6. The osteogenic differentiation of hASCs was investigated by von Kossa, Alizarin red and ALP cellular staining7. The marker of osteogenic differentiation osteopontin, osteocalcin, and ALP was semi-quantified by RT-PCR, and comparison of their expression was made8. The correlation analysis was made among OC,OPN and ALP expressionResults:1. Under SEM, BC membrane presented as an ultrastructural network with nano-scale characteristics.2. hASCs was fibroblast-shaped on the BC membrane, and formed a single layer by9th day.3. The adhesion of hASCs with BC membrane was tight under SEM and frozen section/HE staining.4. hASCs has a good proliferation curve on BC membrane.5. In the process of osteogenic differentiation, hASCs underwent a sequential morphological changes from spindle-like to multishaped, and opaque mineralized nodules could be observed.6. In the osteogenic group, cellular von Kossa staining on plastic plate and BC membrane were both positive,with obvious dark mineralized materials, and negative in non-osteogenic group.7. In the osteogenic group, cellular Alizarin red staining on plastic plate and BC membrane were both positive,with obviously orange-reddish stained cells, and negative in non-osteogenic group.8. In the osteogenic group, cellular ALP staining on plastic plate and BC membrane were both positive,with obviously black-blue stained cytoplasm, and also slightly positive in non-osteogenic group.9. RT-PCR revealed ALP was positively expressed in all groups by2nd and4th week, and was significantly higher in osteogenic groups than non-osteogenic ones; OC and OPN was not markedly expressed by2nd week, but OPN had good expression by4th week, while OC had a weak expression.Conclusions:1. BC has a unique nano-scale ultrastructure. hASCs proliferate and adhere well on its surface, which is suggestive of a good biocompatibility.2. hASCs could undergo a successful osteogenic differentiation on plastic plate using ascorbate, β-glycerophosphate, and dexamethasone.3. hASCs could undergo a successful osteogenic differentiation on BC membrane using ascorbate, β-glycerophosphate, and dexamethasone.4. In the process of osteogenic differentiation, hASCs undergo a sequential morphological changes.5. In the process of osteogenic differentiation, ALP,OC and OPN display a characteristic temporal pattern.6. In the process of osteogenic differentiation, osteogenic markers OC, OPN and ALP has a good correlation with each other in terms of mRNA expression.7. MEDLINE search reveals no report of study on hASCs and BC.