The Combined Effects Of Fluid Shear Stress And Materials Surface Chemistries On The Early Behaviors Of Osteoblasts

Bone damages or even bone defects are often happened due to traffic accidents, sports, aging, reproductive deficiency, or diseases. One potential solution to repair the damaged bones is bone tissue engineering. Seed cells and scaffolds are two key components of tissue engineering. For bone tissue engineering, osteoblasts are typical seed cells. Scaffolds, acting as an artificial extracellular matrix, provide physical support and chemical environment for osteoblasts. Previous studies demonstrated that scaffolds can alter the physiological function of osteoblasts. On the other hand, osteoblasts are stress-sensitive cells so that appropriate mechanical stimuli are required for formation of normal bone tissue. In brief, scaffolds and mechanical stimuli are crucial components for in vitro bioreactor of osteoblasts, both of which have to orchestrate to promise the success of bone tissue engineering. Accordingly, understanding the combined effect of scaffolds and mechanical stimuli may potentially provide guidance for optimal design/selection of scaffold materials and bioreactors so as to realize bone tissue engineering.However, few studies have been focused on the combined effects of scaffolds and mechanical stimuli on osteoblasts, although the effects of scaffold alone or mechanical stimuli alone have been paid wide attentions. Since scaffolds surface chemistry is one of the most important properties of scaffolds, fluid shear stress(FSS) is the main mechanical stimuli in bone tissues, the aim of this study is to investigate the effects of the combined stimuli of materials surface chemistry and FSS on the early responses of osteoblasts. The release of FSS-sensitive chemical factors such as ATP, NO and PGE2, proliferation represented by proliferation index(PI) and differentiation represented by ALP activity were detected to indicate the early osteoblastic responses. Furthermore, it has been verified that focal adhesions and cytoskeletons are both involved in the regulation of osteoblasts responses by surface chemistry alone and FSS alone. Therefore, the other aim of this study is to clarify the potential mechanism by which the combined stimuli regulate osteoblastic responses through observing the focal adhesions and cytoskeletons of osteoblasts before and after the combined stimuli. The main works and conclusions are listed as follows.(1) Various surface chemistries(OH, NH2, CH3) were first prepared on tissue culture glass slide(Glass) by using silanization-based self-assemble monolayer technology. Static water contact angles and X-ray photoelectron spectroscopy(XPS) results verified the successful preparation of OH, NH2, CH3 surfaces. Secondly, a parallel plate flow chamber was constructed to provide FSS. Finally, osteoblasts were seeded on the slides of various surface chemistries, which were then inserted into the flow chamber and suffered from FSS stimuli, providing the platform for the combined stimuli of surface chemistry and FSS.(2) The effects of surface chemistries alone on osteoblasts were investigated. The obtained results are used as controls for the combined stimuli. Osteoblasts on NH2 surfaces were more spread and generated more intense F-atin stress fibers and larger focal adhesions compared to CH3 and OH surfaces, demonstrating a surface-chemistry dependence of NH2(> Glass) > OH ≈ CH3. Compared to Glass surfaces, NH2 obviously promoted whereas CH3 and OH surfaces obviously inhibited the early proliferation and differentiation of osteoblasts. With respect to the release of chemical factors, surface chemistries had no significant effects on the release of both ATP and NO, yet affected the release of PGE2.(3) The effects of the combined stimuli(various surface chemistries and various FSS magnitudes) on osteoblasts were investigated.① The combined stimuli of low FSS(5 dynes/cm2, lower than physiological FSS, labeled as LFSS) and surface chemistries: Compared with the stimuli of surface chemistry alone, LFSS markedly increased the releases of ATP, NO and PGE2, and the osteoblasts proliferation, whereas NH2 and Glass had almost no effects; LFSS promoted osteoblasts orientation along FSS or altered cell aspect ratio on CH3 and OH surfaces, yet produced no changes on NH2 and Glass surfaces. These results, together with the F-actin and focal adhesions formation before LFSS stimuli(that is, receiving surface chemistry stimuli alone), suggest the possible mechanism for the combined stimuli of LFSS and surface chemistry is related to F-actin and focal adhesions formation. Specifically, the surface chemistry first regulates the F-actin and focal adhesions formation, which then lead to high sensitivity of osteoblasts to FSS on CH3 and OH surfaces yet low sensitivity on NH2 surfaces. From the viewpoint of osteoblast sensitivity to FSS, CH3 and OH surfaces are suitable scaffold surface chemistries when LFSS is provided.② The combined stimuli of physiological FSS(12 dynes/cm2, a proved optimum physiological FSS, labeled as PFSS) and surface chemistries: Compared with the stimuli of surface chemistry alone, PFSS obviously increased ATP, NO and PGE2 releases and osteoblasts proliferation on all surfaces, and demonstrated a surface-chemistry dependence of NH2-PFSS(> Glass-PFSS) > OH-PFSS ≈ CH3-PFSS; the osteoblast orientation and cell aspect ratios were altered as well. These results, together with the F-actin and focal adhesions formation before PFSS stimuli(that is, receiving surface chemistry stimuli alone), indicate the key roles of F-actin and focal adhesions formation in regulating the effects of the combined stimuli on osteoblasts responses. For a bioreactor where PFSS is provided, NH2 is the optimum scaffold surface chemistry.③ The combined stimuli of high FSS(20 dynes/cm2, higher than the proved optimum physiological FSS, labeled as HFSS) and surface chemistries: HFSS obviously detached or even ruptured osteoblasts on CH3 and OH surfaces. On contrast, the osteoblasts on NH2 surfaces could resist HFSS and obviously oriented along HFSS. Compared with the stimuli of surface chemistry alone, HFSS significantly promoted the releases of ATP, NO and PGE2, and osteoblasts proliferation. These results suggest that CH3, OH or CH3/OH mixture are not a suitable scaffold surface chemistry when HFSS is provided; instead, NH2 is a better choice.(4) The effects of the combined stimuli(various surface chemistries and various FSS magnitudes) on osteoblasts were systematically analyzed. To clarify the mechanism of the combined stimuli, F-actins were disrupted or/and focal adhesion formations were inhibited, and then the effects of the combined stimuli of PFSS and surface chemistry on osteoblasts were investigated again.① Three concepts relating to FSS, responding threshold, destroying threshold, and optimum threshold were introduced in order to have a clear analysis of the combined effects of various surface chemistries and various FSS magnitudes on osteoblasts. Responding threshold represents the minimum FSS magnitude inducing osteoblast response, destroying threshold represents the minimum FSS magnitude leading to osteoblasts detachment or membrane rupture, and optimum threshold is the FSS magnitude inducing the optimum cell responses, which lies between the responding threshold and the destroying threshold. Compared to Glass surfaces, CH3 and OH down-regulated yet NH2 up-regulated the responding threshold and the destroying threshold of osteoblasts. The osteoblasts of CH3 and OH surfaces should have a responding threshold of < 5 dynes/cm2 and a destroying threshold between 12 dynes/cm2 and 20 dynes/cm2, while the osteoblasts on NH2 surfaces have a responding threshold between 5 dynes/cm2 and 12 dynes/cm2, and a destroying threshold of > 20 dynes/cm2. The different responding thresholds and destroying thresholds on various surface chemistries may explain the observed osteoblast responses upon the combined stimuli of various surface chemistries and various FSS magnitudes. Moreover, the responding thresholds and destroying thresholds depend on the F-actin and focal adhesions.② When focal adhesion formations were inhibited by using free RGDS peptides and then PFSS was applied, PGE2 releases dramatically dropped and lost its surface-chemistry dependence, while ATP and NO releases slightly decreased. This result is consistent with the proved opinion that PGE2 release relies more on focal adhesion formations.③ When F-actins were disrupted by using cytochalasin B and then PFSS was applied, NO releases dramatically dropped and lost its surface-chemistry dependence, while ATP and PGE2 releases slightly decreased. This result is consistent with the proved opinion that NO release requires intact cytoskeleton.④ When both the focal adhesion formations were inhibited and F-actins were disrupted, the osteoblasts on all surface chemistries released nearly no ATP, NO and PGE2.⑤ In all, the damage of both focal adhesion formations and F-actins eliminated the regulation of surface chemistry to PFSS’s effect on osteoblasts; meanwhile, the osteoblasts responses to PFSS were eliminated as well. This result, together with the combined effect of various surface chemistries and FSS magnitudes on osteoblasts, could lead to a conclusion on the mechanism: Surface chemistry can affect the sensitivity(responding threshold) and tolerability(destroying threshold) of osteoblasts to FSS by regulating focal adhesion formations and F-actin cytoskeletons, and further control the osteoblast responses to the combined stimuli of surface chemistry and FSS. Scaffold chemistry should match FSS magnitude in view of the sensitivity and tolerability of osteoblasts to FSS, providing optimum bioreactor and promoting bone formation.