Fibronectin grafting to enhance skin sealing around transcutaneous titanium implant.

Intraosseous transcutaneous amputation prosthesis is a new approach in orthopedic implants that overcomes socket prosthesis problems. Its long-term performance requires a tight skin-implant seal to prevent infections. In this study, fibronectin (Fn), a widely used adhesion protein, was adsorbed or grafted onto titanium alloy. Fn grafting was performed using two different linking arms, dopamine/glutaric anhydride or phosphonate. The characterization of Fn-modified surfaces showed that Fn grating via phosphonate has led to the highest amount of Fn cell-binding site (RGD, arginine, glycine, and aspartate) available on the surface. Interestingly, cell culture studies revealed a strong correlation between the amount of available RGD ligands and cellular behavior, since enhanced proliferation and spreading of fibroblasts were noticed on Fn-grafted surfaces via phosphonate. In addition, an original in vitro mechanical test, inspired from the real situation, to better predict clinical outcomes after implant insertion, has been developed. Tensile test data showed that the adhesion strength of a bio-engineered dermal tissue was significantly higher around Fn-grafted surfaces via phosphonate, as compared to untreated surfaces. This study sheds light on the importance of an appropriate selection of the linking arm to tightly control the spatial conformation of biomolecules on the material surface, and consequently cell interactions at the interface tissue/implant.

Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation.

PURPOSE: To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS: The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-µm-wide/10-µm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen α1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION: The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.

Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

PURPOSE: To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS: The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-microm-wide/10-microm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen alpha1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION: The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.