Cell-Membrane-Inspired Silicone Interfaces that Mitigate Proinflammatory Macrophage Activation and Bacterial Adhesion.

Biofouling on silicone implants causes serious complications such as fibrotic encapsulation, bacterial infection, and implant failure. Here we report the development of antifouling, antibacterial silicones through covalent grafting with a cell-membrane-inspired zwitterionic gel layer composed of 2-methacryolyl phosphorylcholine (MPC). To investigate how substrate properties influence cell adhesion, we cultured human-blood-derived macrophages and Escherichia coli on poly(dimethylsiloxane) (PDMS) and MPC gel surfaces with a range of 0.5-50 kPa in stiffness. Cells attach to glass, tissue culture polystyrene, and PDMS surfaces, but they fail to form stable adhesions on MPC gel surfaces due to their superhydrophilicity and resistance to biofouling. Cytokine secretion assays confirm that MPC gels have a much lower potential to trigger proinflammatory macrophage activation than PDMS. Finally, modification of the PDMS surface with a long-term stable hydrogel layer was achieved by the surface-initiated atom-transfer radical polymerization (SI-ATRP) of MPC and confirmed by the decrease in contact angle from 110 to 20° and the >70% decrease in the attachment of macrophages and bacteria. This study provides new insights into the design of antifouling and antibacterial interfaces to improve the long-term biocompatibility of medical implants.

Exploiting Pharma 4.0 Technologies in the Non-Biological Complex Drugs Manufacturing: Innovations and Implications.

The pharmaceutical industry has entered an era of transformation with the emergence of Pharma 4.0, which leverages cutting-edge technologies in manufacturing processes. These hold tremendous potential for enhancing the overall efficiency, safety, and quality of non-biological complex drugs (NBCDs), a category of pharmaceutical products that pose unique challenges due to their intricate composition and complex manufacturing requirements. This review attempts to provide insight into the application of select Pharma 4.0 technologies, namely machine learning, in silico modeling, and 3D printing, in the manufacturing process of NBCDs. Specifically, it reviews the impact of these tools on NBCDs such as liposomes, polymeric micelles, glatiramer acetate, iron carbohydrate complexes, and nanocrystals. It also addresses regulatory challenges associated with the implementation of these technologies and presents potential future perspectives, highlighting the incorporation of digital twins in this field of research as it seems to be a very promising approach, namely for the optimization of NBCDs manufacturing processes.

Harvesting pre-polarized macrophages using thermo-responsive substrates.

In the cell culture environment macrophages are highly adherent cells. Currently used methods to harvest macrophages have the disadvantage of reducing cell viability and their ability to re-attach after seeding. Although thermo-responsive surfaces have been employed to harvest cell sheets no reports are available to use these to harvest (pre-polarized) macrophages. We show that this method significantly improves the yield of living macrophages and percentage of subsequent cell reattachment, whilst having a minimal effect on the cell phenotype.

Anti-oxidant and immune-modulatory properties of sulfated alginate derivatives on human chondrocytes and macrophages.

Degeneration of articular cartilage represents one of the most common causes of pain and disability in our aging society. Current treatments only address the symptoms of joint disease, but not their underlying causes which include oxidative stress and inflammation in cartilage and surrounding tissues. Sulfated biopolymers that mimic aspects of the native extracellular environment of cartilage are recently gaining interest as a means to slow the inflammatory events responsible for tissue degeneration. Here we show that the natural polysaccharide alginate and particularly its sulfated derivatives have potent anti-oxidant, anti-inflammatory and anti-immunogenic properties in vitro. We found that these polymers exert a free radical scavenging activity in a sulfation-dependent manner. In particular, the sulfation degree of substitution of alginate directly correlated with its ability to scavenge superoxide radicals and to chelate metal ions. We also studied the effect of sulfated alginate on the ability of IL-1ß to stimulate inflammatory genes in human chondrocytes and found decreased expression of the pro-inflammatory markers IL-6 and CXCL8, which inversely correlated with the sulfation degree. Moreover, in studies testing the ability of the alginates to modulate macrophage polarization, we found that they decreased both the gene expression and synthesis of the proinflammatory cytokine TNF-α in human THP-1 macrophages with M1-like phenotype in a sulfation-dependent manner. To conclude, sulfated alginates effectively protect against oxidative stress and inflammation in vitro and are a promising biomaterial to be explored for treatment of osteoarthritis.

Macrophage Polarization by Titanium Dioxide (TiO2) Particles: Size Matters.

Wear particles of total joint replacements may lead to an inflammatory response driven by cells of the monocyte/macrophage lineage. Today, there is a general agreement that the continuous release of wear particles by the implant has a critical impact on periprosthetic osteolysis, which can eventually lead to aseptic loosening of the implant. The focus of this study lay on the determination of the polarization of macrophages (M0) toward the pro-inflammatory M1 phenotype or the anti-inflammatory M2-like phenotype upon exposure to differently sized TiO2 particles. The analysis was done with an in vitro model using THP-1 monocytes. It offers a direct characterization of the polarization profile of the macrophages exposed to nano- (<100 nm, measured hydrodynamic diameter: 518.5 nm) and micro- (<5 µm, measured hydrodynamic diameter: 2213 nm) sized TiO2 particles in different concentrations (4 × 104 -4 × 106 particles/mL). The polarization profile was analyzed by the quantitative assessment of relative gene expression levels as well as by the determination of specific proteins by enzyme linked immunosorbent assay (ELISA). Analysis by qRT-PCR revealed significantly elevated levels of pro-inflammatory markers such as TNF-α and CD197 at the highest concentration of stimulation by the microsized particles. This was confirmed on the protein level in the cytokine expression profile of TNF-α. Furthermore, no significant differences were found for the markers CCL22 and CD206, which are specific for the M2-like phenotype. In contrast, stimulation by nanoparticles did not induce macrophage polarization toward M1 or M2-like phenotype in any applied concentration. We conclude that the size of the particle is a determinant factor in driving the biological response of macrophages and an increased understanding of this relationship may potentially guide the design of new biomaterials.

The influence of nanostructured features on bacterial adhesion and bone cell functions on severely shot peened 316L stainless steel.

Substrate grain structure and topography play major roles in mediating cell and bacteria activities. Severe plastic deformation techniques, known as efficient metal-forming and grain refining processes, provide the treated material with novel mechanical properties and can be adopted to modify nanoscale surface characteristics, possibly affecting interactions with the biological environment. This in vitro study evaluates the capability of severe shot peening, based on severe plastic deformation, to modulate the interactions of nanocrystallized metallic biomaterials with cells and bacteria. The treated 316L stainless steel surfaces were first investigated in terms of surface topography, grain size, hardness, wettability and residual stresses. The effects of the induced surface modifications were then separately studied in terms of cell morphology, adhesion and proliferation of primary human osteoblasts (bone forming cells) as well as the adhesion of multiple bacteria strains, specifically Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and ampicillin-resistant Escherichia coli. The results indicated a significant enhancement in surface work hardening and compressive residual stresses, maintenance of osteoblast adhesion and proliferation as well as a remarkable decrease in the adhesion and growth of gram-positive bacteria (S. aureus and S. epidermidis) compared to non-treated and conventionally shot peened samples. Impressively, the decrease in bacteria adhesion and growth was achieved without the use of antibiotics, for which bacteria can develop a resistance towards anyway. By slightly grinding the surface of severe shot peened samples to remove differences in nanoscale surface roughness, the effects of varying substrate grain size were separated from those of varying surface roughness. The expression of vinculin focal adhesions from osteoblasts was found to be singularly and inversely related to grain size, whereas the attachment of gram-positive bacteria (S. aureus and S. epidermidis) decreased with increasing nanoscale surface roughness, and was not affected by grain refinement. Ultimately, this study demonstrated the advantages of the proposed shot peening treatment to produce multifunctional 316L stainless steel materials for improved implant functions without necessitating the use of drugs.

Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a magneto-mechanically actuated fibrous scaffold.

The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs.

New poly(epsilon-caprolactone)/chitosan blend fibers for tissue engineering applications.

This study reports for the first time on the production of poly(epsilon-caprolactone)/chitosan blend fibers for future application as tissue engineering scaffolds. Fibers of chitosan and poly(epsilon-caprolactone) were prepared by wet spinning from blend solutions, using a formic acid/acetone 70:30vol.% mixture as common solvent and methanol as coagulant. By this method, blend fibers with a wide compositional range and controllable diameters could be produced. Scanning electron microscopy shows the existence of roughness and porosity at the micron level scale in the blend fiber surface that could be potentially advantageous for cell attachment. Studies were also conducted using both conventional and innovative techniques to evaluate compatibility between the polymers, including FTIR imaging and investigation of the glass transition of chitosan using dynamic mechanical analysis on samples with controlled swelling. The data suggest that a certain degree of interaction exists, although it does not seem to be a result of chemical interaction. The designed fibers could be potentially used for the development of scaffolds for tissue engineering applications.