Abstract
Introduction and Objective
The choice of appropriate characteristics is crucial to favor a firm bonding between orthopedic implants and the host bone and to permit bone regeneration. In particular, the morphology and composition of the biointerface plays a crucial role in orchestrating precise cellular responses. Here, to modulate the biointerface, we propose new biomimetic coatings, having multi-scale nano- to micro- morphological cues and a composition mimicking the mineral phase of bone.
Materials and Methods
Films on various substrates are obtained by Ionized Jet Deposition (IJD), by ablation of biogenic apatite and annealing at 400°C for 1 hour. Films are proposed for functionalization of metallic implants, but application to heat sensitive porous (3D printed) substrates is also shown, as it permits to further boost biomimicry (by addition of collagen/gelatin), thus reproducing the architecture of cancellous bone. In IJD, coatings thickness can be selected by tuning deposition duration. Here, a 450 nm thickness is selected based on preliminary results. Micro-rough titanium alloy (Ti6Al4V) disks (roughness 5 μm) are used as a substrate for the deposition and as a control. The coatings are characterized in terms of composition (GI-XRD, EDS, FT-IR microscopy), morphology (FEG-SEM, AFM, data processing by ImageJ), mechanical properties (micro-scratch test) and dissolution profile in medium (pH 7.4, FEG-SEM). Then, their behavior is characterized in vitro (human bone marrow-derived mesenchymal stromal cells - hMSCs), by studying cells early adhesion (focal adhesion by vinculin staining), viability (Alamar Blue), morphology (SEM) and differentiation (expression of RUNX2, ALPL, SPARC and COL1A1, BMP2, BGLAP, osteocalcin, alkaline phosphatase, collagen type I) at 3, 7 and 14 days.
Results
Films exhibit a biomimetic composition, as they are constituted by a nanocrystalline multi-doped carbonated hydroxyapatite. EDS indicates the presence of trace ions sodium (0,11 ± 0,02 wt%) and magnesium (0,47 ± 0,05 wt%), uniformly distributed in the coating in a percentage close to native bone. These ion-substitutions are crucial, as each ion modifies apatite solubility and ion-release in the peri-implant environment and has important biological role. Films have a high adhesion to the substrates and a suitable dissolution profile. The morphology is highly rough, as films are composed by nanosized grains (minimum diameter 40 nm) aggregated in multi-scale clusters (diameter range: 100 nm-2 μm). Morphology of the aggregates can be tuned by selecting deposition duration and also depends on the morphology, roughness and composition of the substrate. Because of the nanoscale thickness of the films, they do not alter the microscale features of the implants. For fibrous substrates, films grow onto the fibers surface, with no pore occlusion or damage to substrate composition. Coatings do not alter the metabolic activity of MSCs but influence their early adhesion, morphology and differentiation. More in detail, MSCs on coated disks show a branched shape, while those on the controls show a more spindle and elongated morphology. Coatings increase hMSCs early adhesion, as a higher density and a greater area of focal adhesions are observed at 24 hours. Finally, they can trigger a signaling pathway that promotes the osteogenic differentiation of hMSCs, as confirmed by quantification of osteocalcin, alkaline phosphatase and collagen, even in the absence of osteogenesis-inducing factors.
Conclusions
The topographical and chemical cues of the biomimetic nanostructured coating are perceived by hMCSs, showing that combining morphological and biomimetic cues is a promising route for the development of cells-instructive biomaterials for orthopedics. In vivo tests on rabbit models are in progress.
