Helena P. Felgueiras

Poly(NaSS) Functionalization Modulates the Conformation of Fibronectin and Collagen Type I To Enhance Osteoblastic Cell Attachment onto Ti6Al4V

Functionalization of surfaces with poly(sodium styrenesulfonate) (poly(NaSS)) has recently been found to enhance osteointegration of implantable materials. Radical polymerization of poly(NaSS) on titanium (Ti)-based substrates has been used to improve their long-term performance by preventing fibrosis and consequently implant loosening. However, the influence of the sulfonate groups on the early cell behavior and the associated molecular phenomena remains to be understood. In this work, we used quartz crystal microbalance with dissipation (QCM-D) to elucidate the role of poly(NaSS) in enhancing osteoblastic cell attachment. This was measured by following the cell attachment using the MC3T3-E1 cell line, on fetal bovine serum (FBS) preadsorbed surfaces and on substrates adsorbed with a series of relevant proteins, bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I). Comparison of the performance of poly(NaSS) with other clinically important substrates such as Ti alloy Ti6Al4V, gold, and poly(desamino-tyrosyl-tyrosine ethyl ester carbonate) (poly(DTEc)) indicates poly(NaSS) to be a superior substrate for MC3T3-E1 cells attachment. This attachment was found to be integrin mediated in the presence of Fn and Col I. Antibodies specific to the RGD peptide and the N- and C-terminal HB-binding domains reacted more intensively with Fn adsorbed on poly(NaSS). Fn adapts a conformation favorable to RGD mediated cell attachment when adsorbed onto poly(NaSS).

Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V

UNLABELLED This study is focused on understanding the underlying mechanisms involved in the improved in vitro and in vivo responses of osteoblasts on poly(sodium styrene sulfonate) (poly(NaSS)) functionalized Ti6Al4V surfaces. We probed the contribution of cell-adhesive glycoproteins fibronectin (Fn) and vitronectin (Vn) in the initial adhesion of MC3T3-E1 osteoblastic cells to poly(NaSS) functionalized and control Ti6Al4V surfaces. Firstly, culture media containing serum depleted of Fn and Vn (DD) were used to establish the contribution of Fn and Vn in the adhesion and spreading of cells on poly(NaSS) grafted and control surfaces. Compared to ungrafted surfaces, poly(NaSS) grafted surfaces enhanced the levels of cell adhesion, cell spreading and the formation of intracellular actin cytoskeleton and focal contacts in serum treatments where Fn or Vn were present (FBS, DD+Fn, DD+Vn). Cell responses to Fn were more significant than to Vn. Secondly, blocking Fn and Vn integrin receptors using antibodies to α5β1 (Fn) and αvβ1 (Vn) showed that adhesion of cells to poly(NaSS) grafted surfaces principally involved the Fn integrin receptor α5β1. Thirdly, blocking of the heparin and cell-binding regions of Fn molecule (RGD, C-HB, N-HB) showed that grafting with poly(NaSS) altered the conformation of Fn. Together these outcomes explained why the presence of sulfonate (SO3(-)) groups grafted on the Ti6Al4V surface enhanced the early cell adhesion and spreading processes which determine clinical success for applications that require osseointegration. STATEMENT OF SIGNIFICANCE This study is devoted to the basic analysis of the mechanism at the origin of the improved in vitro and in vivo osteoblast cell responses exhibited by poly(sodium styrene sulfonate) (poly(NaSS)) functionalized Ti6Al4V surfaces. The aim was to probe the contribution of cell adhesive glycoproteins fibronectin and vitronectin in the initial adhesion of MC3T3-E1 osteoblastic cells to poly(NaSS) functionalized Ti6Al4V surfaces. The outcomes of this research explained why the presence of SO3(-) (sulfonate) groups grafted on the Ti6Al4V surface enhanced the early cell adhesion and spreading processes which determine clinical success for applications that require osseointegration. This work is a step further in the research of poly(NaSS), a very promising bioactive polymer with potential to the orthopedic and dental fields.

The osteogenic differentiation improvement of human mesenchymal stem cells on titanium grafted with polyNaSS bioactive polymer.

Osseointegration of metallic implants used in orthopedic surgery requires that osteoprogenitor cells attach and adhere to the surface, then proliferate, differentiate into osteoblasts, and finally produce mineralized matrix. Because the ability of progenitor cells to attach to a scaffold surface during early stages is important in the development of new tissue structures, we developed in our laboratory, a strategy involving grafting of implants with a polymer of sodium styrene sulfonate (polyNaSS) used as a scaffold which enables human mesenchymal stem cells (hMSCs) interactions. In the present study, we investigated the cellular response of hMSCs to polyNaSS surfaces of titanium (Ti). In particular, cell proliferation, cell viability, cell differentiation, and cell spreading were evaluated. Results showed that cell proliferation and cell viability did not differ with any statistical significance between modified and unmodified Ti surfaces. Interestingly, culture of MSCs on polyNaSS surfaces resulted in a significant increase of cell spreading and cell differentiation compared with the other tested surfaces. These results suggest that titanium surface grafted with polyNaSS is a suitable scaffold for bone tissue engineering.

Biotribocorrosion (tribo-electrochemical) characterization of anodized titanium biomaterial containing calcium and phosphorus before and after osteoblastic cell culture

The purpose of this study was to investigate the relationship between the osteoblastic cells behavior and biotribocorrosion phenomena on bioactive titanium (Ti). Ti substrates submitted to bioactive anodic oxidation and etching treatments were cultured up to 28 days with MG63 osteoblast-like cells. Important parameters of in vitro bone-like tissue formation were assessed. Although no major differences were observed between the surfaces topography (both rough) and wettability (both hydrophobic), a significant increase in cell attachment and differentiation was detected on the anodized substrates as product of favorable surface morphology and chemical composition. Alkaline phosphatase production has increased (≈20 nmol/min/mg of protein) on the anodized materials, while phosphate concentration has reached the double of the etched material and calcium production increased (over 20 µg/mL). The mechanical and biological stability of the anodic surfaces were also put to test through biotribocorrosion sliding solicitations, putting in evidence the resistance of the anodic layer and the cells capacity of regeneration after implant degradation. The Ti osteointegration abilities were also confirmed by the development of strong cell-biomaterial bonds at the interface, on both substrates. By combining the biological and mechanical results, the anodized Ti can be considered a viable option for dentistry.

Competitive Adsorption of Plasma Proteins Using a Quartz Crystal Microbalance

Proteins that get adsorbed onto the surfaces of biomaterials immediately upon their implantation mediate the interactions between the material and the environment. This process, in which proteins in a complex mixture compete for adsorption sites on the surface, is determined by the physicochemical interactions at the interface. Competitive adsorption of bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I), sequentially and from mixtures, was investigated so as to understand the performances of different surfaces used in biomedical applications. A quartz crystal microbalance with dissipation was used to monitor the adsorption of these proteins onto two materials used in functional bone replacement, a titanium alloy (Ti6Al4V) and Ti6Al4V physisorbed with poly(sodium styrenesulfonate) [poly(NaSS)], and three controls, gold, poly(desaminotyrosyltyrosine ethyl ester carbonate) [poly(DTEc)], and polystyrene (PS). In experiments with individual proteins, the adsorption was the highest with Fn and Col I and the least with BSA. Also, protein adsorption was the highest on poly(NaSS) and Ti6Al4V and the least on poly(DTEc). In sequential adsorption experiments, protein exchange was observed in BSA + Fn, Fn + Col I, and BSA + Col I sequences but not in Fn + BSA and Col I + BSA because of the lower affinity of BSA to surfaces relative to Fn and Col I. Protein adsorption was the highest with Col I + Fn on hydrophobic surfaces. In experiments with protein mixtures, with BSA & Fn, Fn appears to be preferentially adsorbed; with Fn & Col I, both proteins were adsorbed, probably as multilayers; and with Col I & BSA, the total amount of protein was the highest, greater than that in sequential and individual adsorption of the two proteins, probably because of the formation of BSA and Col I complexes. Protein conformational changes induced by the adsorbing surfaces, protein-protein interactions, and affinities of proteins appear to be the important factors that govern competitive adsorption. The findings reported here will be useful in understanding the host response to surfaces used for implants.

Bone tissue response induced by bioactive polymer functionalized Ti6Al4V surfaces: In vitro and in vivo study

Ti6Al4V is commonly used for orthopedic applications. This study was designed to test the potentially added benefit of Ti6Al4V functionalized with a bioactive polymer poly(sodium styrene sulfonate) both in vitro and in vivo. Cell-based assays with MC3T3-E1 osteoblast-like cells were used to measure the cell adhesion strength, cell spreading, focal contact formation, cell differentiation and the mineralization of extracellular matrix on grafted and ungrafted Ti6Al4V discs in combination with FBS and collagen type I. Bone morphogenetic protein-2 (BMP-2) was also included in the cell differentiation assay. Results showed that the grafted surface combined with collagen I gave superior levels in every parameter tested with cell-based assays and was almost equivalent to BMP-2 for cell differentiation. In vivo testing was conducted in rabbits (n=42) with cylinders of grafted and ungrafted Ti6Al4V implanted in defects made to the femoral and lateral condyles and animals that were maintained to 1, 3 and 12months. Hydroxyapatite coated Ti6Al4V cylinders were included as a clinical reference control. Osseointegration was assessed post-mortem using histomorphometric analysis conducted on resin sections of explanted undecalcified bone. Two histomorphometric parameters, that of bone-to-implant contact and the bone area, were analyzed by a trained observer blinded to sample identity. Results showed osseointegration on grafted Ti6Al4V was marginally better than both ungrafted and hydroxyapatite coated Ti6Al4V. Overall, the study found that the grafted Ti6Al4V significantly promoted all aspects of osteogenesis tested in vitro and, although in vivo outcomes were less compelling, histomorphometry showed osseointegration of grafted Ti6Al4V implants was equivalent or better than controls.

Competitive Adsorption of Albumin, Fibronectin and Collagen Type I on Different Biomaterial Surfaces: A QCM-D Study

Adsorption of several proteins, individually and in a mixture, onto substrates was investigated. Five biomaterial surfaces were selected based on their functionality and/or hydrophilicity: Ti6Al4V, Ti6Al4V + poly(sodium styrene sulfonate) (poly(NaSS)), gold, poly(desamino tyrosyltyrosine ethyl ester carbonate) (poly(DTE)), polystyrene (PS). Their ability to interact with BSA, Fn and Col I was studied using a QCM-D technique.