Biological Effects Of Fluid Flow On Bone Cells And Application For Bone Tissue Engineering

Mechanical loading is an important regulator of bone metabolism. Increasedmechanical loading stimulates bone formation and suppresses bone resorption,leading to an increase in bone mass. Conversely, skeletal unloading results inincreased bone resorption, decreased bone mineral density, decreased boneformation, decreased bone growth and strength, and reduced mineralization.Mechanical loading produces strains in the mineralized matrix of bone that arethought to generate interstitial fluid flow through the lacunar/canalicular spaces.This fluid flow exerts a shear stress at surfaces of osteoblasts and osteocyteslining these spaces,which influences bone cell metabolism and boneadaptation.To study of the biological fluid flow on bone cells will not only contribute tostudy on the bone development and adaptation, but to utilize mimicking the IFFalso could be potential useful for bone tissue engineering. In present study, westudied the biological responses of osteoblast-like cells in different maturationstages under shear flow. To mimic microphysical enviroments such as theoscillatory IFF in vivo, a novel oscillatory perfusion system was designed. Itsefficiency was futher evaluated.1. Biological Responses of Osteoblast-like Cells in Different MaturationStages under Shear Flow.This part of research was to sturdy the biological responses of osteoblast-likecells in different maturation stages under shear flow. Shear flow stimulation of4.62dyn·s/cm2 was applied by parallel flow chamber system for 2 hours onosteoblast-like cell MC 3T3-E1 of different maturation stage induced byosteogenic media, and then collect samples for dsDNA content and ALP activityanalysis.The results showed that on 24 hours after stimulation, there was notobvious change of ALP activity with non-osteogenic inducing group, anincrease was observed in the group of 4 days of differentiation, while andecrease in the group of 8 days of differentiation. So the biological responses ofosteoblast-like cells in different maturation stages under shear flow is quitedifferent, and this may be an important role in bone growth and reconstruction.2. Oscillatory Perfusion Seeding and Culturing of Osteoblast-like Cells onPorous Beta-Tricalcium Phosphate ScaffoldsBy nutrient transport and mechanical stimulation, perfusion culture systemswith unidirectional flow have been proven effective bioreactors for constructionof tissue engineering bone in vitro. But bone interstitial flow in vivo isoscillatory in nature. Thus, in the present study, a compact perfusion systemwith oscillatory flow was developed, and its seeding and culture effects wereevaluated. Mouse osteoblast-like cells, MC 3T3-E1, were seeded and culturedfor 6 days in large porous ceramic -tricalcium phosphate (?-TCP) scaffolds byeither the static or oscillatory perfusion method. The seeding efficiency, cellproliferation, early osteogenesis, distribution and viability were evaluated.Compared with the static culture, the oscillatory perfusional method showedsignificantly higher seeding efficiency and higher scaffold cellularity in DNAcontent analysis, as well as higher ALP activity after 6 days of culture.Stereomicroscope observation of MTT staining and Hoechst/PI double stainingalso demonstrated homogeneous seeding, proliferation and viability of cellsthroughout in the oscillatory system;by contrast, the static culture showedpolarized seeding and proliferation favoring only the outer and upper surfaces,with only dead cells in central part of the scaffolds. The results demonstratedthat the oscillatory flow condition not only allows a better seeding efficiencyand homogeneity, but also facilitates uniform culture and osteogenicdifferentiation. These results suggested that the oscillatory perfusion systemcould be a simple and effective bioreactor for bone tissue engineering, as wellas a 3D model for biological study of oscillatory flow in bone.