Effects Of Mechanical Pressure On Biological Behavior Of BMSCs Cultured On Titanium Surfaces

Establishment of direct bone-implant connection, known as osseointegration, is considered to be the biological fundamentals for the restoration. Studies have found that Titania nanotube array could simulate the collagen arrangement in natural bone tissue[1], and it’s elastic modulus is similar to natural bone tissue that might possibly attract and induce osteogenic differentiation of bone marrow mesenchymal stem cells around the implant zone[2]. Another study found that hydrostatic pressure enhances cellular viability and improves osteogenic differentiation and maturation of human mesenchymal stem cells[3]; Fluid flow shear stress can promote the osteogenic differentiation of cultured MSCs[4, 5]; Moreover, both cyclic and intermittent hydrostatic compression(IHC) could promote bone development[3, 6, 7]. Unfortunately, there is still no report of cellular response to mechanical stimulus on titanium implants surface. Therefore, it is essential to figure out the dual effect of mechanical force and implant on cellular behavior. In our study, the primary rat mesenchymal stem cells were observed on titania nanotube array fabricated by anodization under different intermittent hydrostatic compression, aiming to simulate a more realistic condition in vitro.PartⅠ Fabrication and surface performance analysis of titanium nanotube array[Objective] Fabricating titanium nanotube array on pure titanium samples.[Methods] Titanium samples were polished using Si C sandpaper, titanium nanotube array were Fabricated via Anodizing and observed by FE-SEM.[Results] Anodization of pure titanium samples formed Ti O2 nanotube array on the surface, The Ti O2 nanotube array has consistent pipe diameter and uniform distribution.[Conclusion] Titania nanotube array can be formed by anodizing of pure titanium samples.PartⅡ Cultivation and characterization of BMSCs[Objective]To isolate and identify the biological characters of rat BMSCs.[Methods]According to the method of whole bone marrow adherent, we isolated BMSCs of rat successfully and observed them by light microscopy. Flow cytometry, MTT, staining tests(alizarin red and oil red O)as well as clone formation assay were used to justify what we obtained were mesenchymal stem cells.[Results]Rat BMSCs under optical microscope were fusiform, triangular or polygonal and positively expressed MSC markers CD29(97.75%),negative expressed hematopoietic cells markers CD45(1.86%) and endothelial cells specificity molecular phenotypic CD31(1.07%);All the staining tests proved that cells which we isolated from rat have the ability of multiple differentiation potential.[Conclusion] We isolated BMSCs of rat successfully by the method of whole bone marrow adherent, The obtained BMSCs have the ability of self-renewal and muti-potential differentiation potential.PartⅢ Effects of mechanical pressure on biological behavior of BMSCs cultured on titanium surfaces[Objective]To simulate a more realistic condition in vitro and investigate the effects of mechanical pressure on biological behavior of BMSCs cultured on titanium surfaces.[Methods]Pure titanium samples were divided into Polished Ti(PT) and Ti O2 nanotube array(NT). BMSCs were obtained from SD rats and Inoculated on titanium samples, Then the BMSCs were treated by different hydrostatic pressure(0kpa、13kpa、68kpa、120kpa) for 1 h/d, CCK-8 method is used to determine cell activity for 1,4,7d respectively; Fluorescence staining and FE-SEM are used to show the cell skeleton and morphology after the cells were cultured for 3d, Flow cytometry is used to detect cell apoptosis, ALP activity is measured by commercial kit after the cells were cultured for 7d. collagen secretion as well as extracellular matrix mineralization were stained by Sirius red and Alizarin after the cells were cultured for 14 d.[Results](1)Loading hydrostatic pressure(13kpa、68kpa、120kpa) to BMSCs did not result in significant cell apoptosis.(2) Compared with 0kpa group, hydrostatic pressure may promote the proliferation of BMSCs after the cells were cultured for 4d(p < 0.05); the actin filaments became denser and thicker, The shape of cells on all surface was more elongated and pseudopodium was also become larger after loading hydrostatic pressure. nanotopography collaborate with hydrostatic pressure for 7-14 d, ALP activity, collagen secretion as well as extracellular matrix mineralization was enhanced but proliferation of BMSCs were decreased(p < 0.05).[Conclusion]mechanical pressure and substrate nanotopography may synergistically effect the proliferation, cytoskeleton, morphology as well as differentiation of BMSCs.