Influence of nanosecond laser surface patterning on dental 3Y-TZP: Effects on the topography, hydrothermal degradation and cell response.

OBJECTIVES: Laser surface micropatterning of dental-grade zirconia (3Y-TZP) was explored with the objective of providing defined linear patterns capable of guiding bone-cell response. METHODS: A nanosecond (ns-) laser was employed to fabricate microgrooves on the surface of 3Y-TZP discs, yielding three different groove periodicities (i.e., 30, 50 and 100 µm). The resulting topography and surface damage were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). X-Ray diffraction (XRD) and Raman spectroscopy techniques were employed to assess the hydrothermal degradation resistance of the modified topographies. Preliminary biological studies were conducted to evaluate adhesion (6 h) of human mesenchymal stem cells (hMSC) to the patterns in terms of cell number and morphology. Finally, Staphylococcus aureus adhesion (4 h) to the microgrooves was investigated. RESULTS: The surface analysis showed grooves of approximately 1.8 µm height that exhibited surface damage in the form of pile-up at the edge of the microgrooves, microcracks and cavities. Accelerated aging tests revealed a slight decrease of the hydrothermal degradation resistance after laser patterning, and the Raman mapping showed the presence of monoclinic phase heterogeneously distributed along the patterned surfaces. An increase of the hMSC area was identified on all the microgrooved surfaces, although only the 50 µm periodicity, which is closer to the cell size, significantly favored cell elongation and alignment along the grooves. A decrease in Staphylococcus aureus adhesion was observed on the investigated micropatterns. SIGNIFICANCE: The study suggests that linear microgrooves of 50 µm periodicity may help in promoting hMSC adhesion and alignment, while reducing bacterial cell attachment.

Peptidic biofunctionalization of laser patterned dental zirconia: A biochemical-topographical approach.

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 µm. A platform containing the cell-adhesive RGD and the osteogenic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron microscopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interestingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After biofunctionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 µm, the largest effects were observed from the biomolecule, which favored upregulation of several genes related to osteoblastic differentiation in all the surfaces.

Control of stem cell response and bone growth on biomaterials by fully non-peptidic integrin selective ligands.

In this communication we report that anchoring αvß3 or α5ß1 integrin-selective RGD peptidomimetics to titanium efficiently tunes mesenchymal stem cell response in vitro and bone growth in rat calvarial defects. Our results demonstrate that this molecular chemistry-derived approach could be successful to engineer instructive coatings for orthopedic applications.

Tuning Mesenchymal Stem Cell Response onto Titanium-Niobium-Hafnium Alloy by Recombinant Fibronectin Fragments.

Since metallic biomaterials used for bone replacement possess low bioactivity, the use of cell adhesive moieties is a common strategy to improve cellular response onto these surfaces. In recent years, the use of recombinant proteins has emerged as an alternative to native proteins and short peptides owing to the fact that they retain the biological potency of native proteins, while improving their stability. In the present study, we investigated the biological effect of two different recombinant fragments of fibronectin, spanning the 8-10th and 12-14th type III repeats, covalently attached to a new TiNbHf alloy using APTES silanization. The fragments were studied separately and mixed at different concentrations and compared to a linear RGD, a cyclic RGD and the full-length fibronectin protein. Cell culture studies using rat mesenchymal stem cells demonstrated that low to medium concentrations (30% and 50%) of type III 8-10th fragment mixed with type III 12-14th fragment stimulated cell spreading and proliferation compared to RGD peptides and the fragments separately. On the other hand, type III 12-14th fragment alone or mixed at low volume percentages ≤50% with type III 8-10th fragment increased alkaline phosphatase levels compared to the other molecules. These results are significant for the understanding of the role of fibronectin recombinant fragments in cell responses and thus to design bioactive coatings for biomedical applications.

Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion.

Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8-10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8-10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials.