Combined Effects Of Low Fluid Shear Stress And Substrate Chemistry On The Proliferation And Differentiation Of Osteoblasts

Seed cell is a key component of bone tissue engineering. Modulation of seed cells’physiological functions to enhance bone formation is essentially important. Thephysiological functions of seed cells are governed by their surroundingmicroenvironment, including chemical and biophysical ones, which orchestrate toregulate cellular functions. Accordingly, understanding the synergistic effect ofchemical and biophysical microenvironment may potentially provide guidance fordesign and manipulate artificial microenvironment to realize bone tissue engineering.The aim of this study is to investigate the combined effect of fluid shear stress （FSS）and substrate chemistry on the cellular reponses of osteoblasts and explore the potentialmechanism.For bone tissue, FSS is one of the important mechanical stimuli in the biophysicalmicroenvironment of osteogenic cells. FSS can be converted into biochemical signal viamechanotransduction components and then provoke a variety of signaling pathways,promoting osteogenesis. Cytoskeleton and focal adhesion are crucial components ofmechanotransduction. On the other hand, bone tissue engineering scaffold, acting as anartificial extracellular matrix, can provide physical support and chemical environmentas well for cells. Previous studies demonstrated that scaffold chemistry can alterthephysiological function of osteogenic cells by regulatingcell adhesion, morphology andmigration. Accordingly, we speculate that substrate chemistry can affect the responsesof osteogenic cells to FSS and further control bone formation.To test our hypothesis and explore the comprehensive impacts of FSS and substratechemistry on osteoblasts behavior, the following works were carried out:â‘ Preparation of substrate surfaces with various ending groups. Substrate surfaceswith various ending groups （-OH, NH₂, CH₃） were grafted on glass slide surfaces byusing self-assembly monolayer （SAM） technology. The successful preparation wasverified via detection of static water contact angles.â‘¡Application of low FSS （5dyn/cm²ï¼‰ on osteoblasts. After primary SD ratosteoblasts cultured on different surfaces reached confluent,5dyn/cm²FSS was loadedfor0h or1h by using a parallel plate flow chamber device, which were labeled FSS-orFSS⁺group, respectively. â‘¢Evaluation of osteoblasts function. The cell proliferation, alkaline phosphatase（ALP） activity, F-actin organization and vinculin distribution, content of fibronectin （Fn）and type I collagen （COL I） of FSS-and FSS⁺osteoblasts were examined by means offlow cytometry, ALP-kit, immunofluorescence technique and western blot, respectively.The major results and conclusions are listed as follows:â‘ Low FSS promotes proliferation and inhibits early differentiation of osteoblasts.Obvious effect of FSS on the extracellular matrix proteins expression was observed atthe initial stage of cell culture, however, this effect disappeared with the extended cellculture.â‘¡The effect of low FSS on osteoblasts function was regulated by substratechemistry.5dyn/cm²FSS increased the S-phase fraction on all surfaces. And theincrease rate of S-phase fraction on OH surfaces compared to the corresponding FSS-controls are higher than on CH₃and NH₂surfaces. ALP activity decreased in responseto5dyn/cm²FSS, and the decrease rate followed the trend of OH> Blank≈CH₃> NH₂.In a brief summary, osteoblasts on OH surface and CH₃surfaces are more sensitive to5dyn/cm²FSS than on NH₂surfaces.â‘¢F-actin organization and focal adhesion may be responsible for the discrepantresponse. Before FSS exposure, osteoblasts on NH₂surfaces were more spread andgenerated larger focal adhesions so that5dyn/cm²FSS is not strong enough to deformthe osteoblasts. After FSS exposure, no obvious change of both F-actin organization andfocal adhesions was observed for osteoblasts on NH₂surfaces while obvious changewas observed on CH₃and OH surfaces. This suggests that substrate chemistrymodulates the sensitivity of osteoblasts to5dyn/cm²FSS via regulating F-actinorganization and focal adhesion.â‘£The content of Fn and COL I in response to5dyn/cm²FSS were dependent onsubstrate chemistry: both Fn and COL I on CH₃surfaces increased while on OHsurfaces Fn increased and COL I decreased. For NH₂surfaces,5dyn/cm²FSS hadnegligible effect on Fn and COL I content.⑤Although F-actin organization and focal adhesion formation demonstrated thatosteoblasts on OH surfaces are more sensitive to5dyn/cm²FSS, COL I expression waslow. This may be explained by the high hydrophilicity of OH surfaces which restrictsthe assembly of soluble COL I into insoluble one. As a result, it is recommended thatthe effect of scaffold chemistry on ECM assembly should be taken into account while designing scaffold chemistry.