The in vitro mimicking of bone microenvironment for the study of pathologies is a challenging field that requires the design of scaffolds with suitable morphological, structural and cytocompatible properties. During last years, 3D in vitro tumour models have been developed to reproduce mechanical, biochemical and structural bone microenvironment elements, allowing cells to behave as in vivo. In this work, gas foamed polyether urethane foams (PUF) and 3D printed thermoplastic polyether urethane (3DP-PU) designed with different patterns are proposed as scaffolds for in vitro model of bone tissue. Surface coatings for a biomimetic behaviour of the 3D scaffold models were also investigated. Morphological, chemico-physical, mechanical properties, and biological in vitro behaviour were investigated.
In conclusion, based on the obtained results, scaffolds with different geometries have been successfully produced. Morphological and structural properties of the scaffolds here presented are suitable for mimicking the bone tissue, in order to produce a 3D in vitro model useful for bone pathologies research.
Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage.
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. 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.Introduction and Objective
Materials and Methods
Calcium phosphates-based coatings have been widely studied to favour a firm bonding between orthopaedic implants and the host bone. To this aim, thin films (thickness below 1 μm) having high adhesion to the substrate and a nanostructured surface texture are desired, capable of boosting platelet, proteins and cells adhesion. In addition, a tunable composition is required to resemble as closely as possible the composition of mineralized tissues and/or to intentionally substitute ions having possible therapeutic functions. The authors demonstrated nanostructured films having high surface roughness and a composition perfectly resembling the deposition target one can be achieved by Ionized Jet Deposition (IJD). Highly adhesive nanostructured coatings were obtained by depositing bone-apatite like thin films by ablation of deproteinized bovine bone, capable of promoting host cells attachment, proliferation and differentiation. Here, biomimetic films are deposited by IJD, using biogenic and synthetic apatite targets. Since IJD deposition can be carried out without heating the substrate, application on heat sensitive polymeric substrate, Biogenic apatite coatings are obtained by deposition of deproteinized bone (bovine, ovine, equine, porcine) and compared to ones of stoichiometry hydroxyapatite (HAp). Coatings composition (FT-IR-ATR, FT-IR microscopy, XRD, EDS) and morphology (SEM, AFM) are tested for deposition onto metallic and 3D-printed polymeric substrates (polyurethane (PU)). Different post-treatment annealing procedures for metallic substrates are compared (350–425°C), to optimize crystallinity. Then, uniformity of substrate coverage and possible damage caused to the polymeric substrate are studied by SEM, DSC and FT-IR microscopy. Biogenic coatings are composed by carbonated HAp (XRD, FT-IR). Trace ions Na+ and Mg2+ are transferred from deposition target to coating. All coatings are nanostructured, composed by nano-sized globular aggregates, of which morphology and dimensions depend on the target characteristics. As-deposited coatings are amorphous, but crystallinity can be tuned by post-treatment annealing. A bone-like crystallinity can be achieved for heating at ≥400°C, also depending on duration. When deposited on 3D-printed PU scaffolds, coatings, owing to sub-micrometric thickness, coat them entirely, without altering their fibre shape and porosity. Obtained biomimetic bone apatite coatings can be deposited onto a variety of metallic and polymeric biomedical devices, thus finding several perspective applications in biomedical field.
In vitro cell interactions were evaluated with human osteoblasts (HOB, 2nd passage) isolated from the trabecular bone of the femoral head of patients undergoing total hip replacement and cultured following the usual procedure. HOB cells (1x105 cells/sample) were kept in contact with the scaffolds for 7 and 14 days. At each time endpoint HOB metabolic activity, intracellular and released ALP were evaluated.
HOB cells grown on scaffold samples showed an increase of metabolic activity from 7 to 14 days. The amount of intracellular ALP increased too, whereas the amount of ALP in the medium was quite low. HOB cells, after 14 days, appeared closely adherent to the scaffolds, with an elongated and flattened shape.