Design of Laser Activated Antimicrobial Porous Tricalcium Phosphate-Hydroxyapatite Scaffolds for Orthopedic Applications.

The field of bone tissue engineering is steadily being improved by novel experimental approaches. Nevertheless, microbial adhesion after scaffold implantation remains a limitation that could lead to the impairment of the regeneration process, or scaffold rejection. The present study introduces a methodology that employs laser-based strategies for the development of antimicrobial interfaces on tricalcium phosphate-hydroxyapatite (TCP-HA) scaffolds. The outer surfaces of the ceramic scaffolds with inner porosity were structured using a femtosecond laser (λ = 800 nm; τ = 70 fs) for developing micropatterns and altering local surface roughness. The pulsed laser deposition of ZnO was used for the subsequent functionalization of both laser-structured and unmodified surfaces. The impact of the fs irradiation was investigated by Raman spectroscopy and X-ray diffraction. The effects of the ZnO-layered ceramic surfaces on initial bacterial adherence were assessed by culturing Staphylococcus aureus on both functionalized and non-functionalized scaffolds. Bacterial metabolic activity and morphology were monitored via the Resazurin assay and microscopic approaches. The presence of ZnO evidently decreased the metabolic activity of bacteria and led to impaired cell morphology. The results from this study have led to the conclusion that the combination of fs laser-structured surface topography and ZnO could yield a potential antimicrobial interface for implants in bone tissue engineering.

Single-Step Process for Titanium Surface Micro- and Nano-Structuring and In Situ Silver Nanoparticles Formation by Ultra-Short Laser Patterning.

Ultra-short laser (USL)-induced surface structuring combined with nanoparticles synthesis by multiphoton photoreduction represents a novel single-step approach for commercially pure titanium (cp-Ti) surface enhancement. Such a combination leads to the formation of distinct topographical features covered by nanoparticles. The USL processing of cp-Ti in an aqueous solution of silver nitrate (AgNO3) induces the formation of micron-sized spikes surmounted by silver nanoparticles (AgNPs). The proposed approach combines the structuring and oxidation of the Ti surface and the synthesis of AgNPs in a one-step process, without the use of additional chemicals or a complex apparatus. Such a process is easy to implement, versatile and sustainable compared to alternative methodologies capable of obtaining comparable results. Antimicrobial surfaces on medical devices (e.g., surgical tools or implants), for which titanium is widely used, can be realized due to the simultaneous presence of AgNPs and micro/nano-structured surface topography. The processed surfaces were examined by means of a scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM) and Raman spectroscopy. The surface morphology and the oxidation, quality and quantity of AgNPs were analyzed in relation to process parameters (laser scanning speed and AgNO3 concentration), as well as the effect of AgNPs on the Raman signal of Titanium oxide.

Optimizing the Surface Structural and Morphological Properties of Silk Thin Films via Ultra-Short Laser Texturing for Creation of Muscle Cell Matrix Model.

Temporary scaffolds that mimic the extracellular matrix's structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity-the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix.

Investigating Potential Effects of Ultra-Short Laser-Textured Porous Poly-ε-Caprolactone Scaffolds on Bacterial Adhesion and Bone Cell Metabolism.

Developing antimicrobial surfaces that combat implant-associated infections while promoting host cell response is a key strategy for improving current therapies for orthopaedic injuries. In this paper, we present the application of ultra-short laser irradiation for patterning the surface of a 3D biodegradable synthetic polymer in order to affect the adhesion and proliferation of bone cells and reject bacterial cells. The surfaces of 3D-printed polycaprolactone (PCL) scaffolds were processed with a femtosecond laser (λ = 800 nm; τ = 130 fs) for the production of patterns resembling microchannels or microprotrusions. MG63 osteoblastic cells, as well as S. aureus and E. coli, were cultured on fs-laser-treated samples. Their attachment, proliferation, and metabolic activity were monitored via colorimetric assays and scanning electron microscopy. The microchannels improved the wettability, stimulating the attachment, spreading, and proliferation of osteoblastic cells. The same topography induced cell-pattern orientation and promoted the expression of alkaline phosphatase in cells growing in an osteogenic medium. The microchannels exerted an inhibitory effect on S. aureus as after 48 h cells appeared shrunk and disrupted. In comparison, E. coli formed an abundant biofilm over both the laser-treated and control samples; however, the film was dense and adhesive on the control PCL but unattached over the microchannels.

Ultra-Short Laser Surface Properties Optimization of Biocompatibility Characteristics of 3D Poly-ε-Caprolactone and Hydroxyapatite Composite Scaffolds.

The use of laser processing for the creation of diverse morphological patterns onto the surface of polymer scaffolds represents a method for overcoming bacterial biofilm formation and inducing enhanced cellular dynamics. We have investigated the influence of ultra-short laser parameters on 3D-printed poly-ε-caprolactone (PCL) and poly-ε-caprolactone/hydroxyapatite (PCL/HA) scaffolds with the aim of creating submicron geometrical features to improve the matrix biocompatibility properties. Specifically, the present research was focused on monitoring the effect of the laser fluence (F) and the number of applied pulses (N) on the morphological, chemical and mechanical properties of the scaffolds. SEM analysis revealed that the femtosecond laser treatment of the scaffolds led to the formation of two distinct surface geometrical patterns, microchannels and single microprotrusions, without triggering collateral damage to the surrounding zones. We found that the microchannel structures favor the hydrophilicity properties. As demonstrated by the computer tomography results, surface roughness of the modified zones increases compared to the non-modified surface, without influencing the mechanical stability of the 3D matrices. The X-ray diffraction analysis confirmed that the laser structuring of the matrices did not lead to a change in the semi-crystalline phase of the PCL. The combinations of two types of geometrical designs-wood pile and snowflake-with laser-induced morphologies in the form of channels and columns are considered for optimizing the conditions for establishing an ideal scaffold, namely, precise dimensional form, mechanical stability, improved cytocompatibility and antibacterial behavior.

Biomimetic Hierarchical Structuring of PLA by Ultra-Short Laser Pulses for Processing of Tissue Engineered Matrices: Study of Cellular and Antibacterial Behavior.

The influence of ultra-short laser modification on the surface morphology and possible chemical alteration of poly-lactic acid (PLA) matrix in respect to the optimization of cellular and antibacterial behavior were investigated in this study. Scanning electron microscopy (SEM) morphological examination of the processed PLA surface showed the formation of diverse hierarchical surface microstructures, generated by irradiation with a range of laser fluences (F) and scanning velocities (V) values. By controlling the laser parameters, diverse surface roughness can be achieved, thus influencing cellular dynamics. This surface feedback can be applied to finely tune and control diverse biomaterial surface properties like wettability, reflectivity, and biomimetics. The triggering of thermal effects, leading to the ejection of material with subsequent solidification and formation of raised rims and 3D-like hollow structures along the processed zones, demonstrated a direct correlation to the wettability of the PLA. A transition from superhydrophobic (θ > 150°) to super hydrophilic (θ < 20°) surfaces can be achieved by the creation of grooves with V = 0.6 mm/s, F = 1.7 J/cm2. The achieved hierarchical architecture affected morphology and thickness of the processed samples which were linked to the nature of ultra-short laser-material interaction effects, namely the precipitation of temperature distribution during material processing can be strongly minimized with ultrashort pulses leading to non-thermal and spatially localized effects that can facilitate volume ablation without collateral thermal damage The obtained modification zones were analyzed employing Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), Energy dispersive X-ray analysis (EDX), and optical profilometer. The modification of the PLA surface resulted in an increased roughness value for treatment with lower velocities (V = 0.6 mm/s). Thus, the substrate gains a 3D-like architecture and forms a natural matrix by microprocessing with V = 0.6 mm/s, F = 1.7 J/cm2, and V = 3.8 mm/s, F = 0.8 J/cm2. The tests performed with Mesenchymal stem cells (MSCs) demonstrated that the ultra-short laser surface modification altered the cell orientation and promoted cell growth. The topographical design was tested also for the effectiveness of bacterial attachment concerning chosen parameters for the creation of an array with defined geometrical patterns.