A Simple and Cost-Effective FeCl3-Catalyzed Functionalization of Cellulose Nanofibrils: Toward Adhesive Nanocomposite Materials for Medical Implants.

In the present work, we explored Lewis acid catalysis, via FeCl3, for the heterogeneous surface functionalization of cellulose nanofibrils (CNFs). This approach, characterized by its simplicity and efficiency, facilitates the amidation of nonactivated carboxylic acids in carboxymethylated cellulose nanofibrils (c-CNF). Following the optimization of reaction conditions, we successfully introduced amine-containing polymers, such as polyethylenimine and Jeffamine, onto nanofibers. This introduction significantly enhanced the physicochemical properties of the CNF-based materials, resulting in improved characteristics such as adhesiveness and thermal stability. Reaction mechanistic investigations suggested that endocyclic oxygen of cellulose finely stabilizes the transition state required for further functionalization. Notably, a nanocomposite, containing CNF and a branched low molecular weight polyethylenimine (CNF-PEI 800), was synthesized using the catalytic reaction. The composite CNF-PEI 800 was thoroughly characterized having in mind its potential application as coating biomaterial for medical implants. The resulting CNF-PEI 800 hydrogel exhibits adhesive properties, which complement the established antibacterial qualities of polyethylenimine. Furthermore, CNF-PEI 800 demonstrates its ability to support the proliferation and differentiation of primary human osteoblasts over a period of 7 days.

Physically and Chemically Crosslinked Hyaluronic Acid-Based Hydrogels Differentially Promote Axonal Outgrowth from Neural Tissue Cultures.

Our aim was to investigate axonal outgrowth from different tissue models on soft biomaterials based on hyaluronic acid (HA). We hypothesized that HA-based hydrogels differentially promote axonal outgrowth from different neural tissues. Spinal cord sliced cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) were maintained on a collagen gel, a physically crosslinked HA-based hydrogel (Healon 5®) and a novel chemically crosslinked HA-based hydrogel, with or without the presence of neurotrophic factors (NF). Time-lapse microscopy was performed after two, five and eight days, where axonal outgrowth was assessed by automated image analysis. Neuroprotection was investigated by PCR. Outgrowth was observed in all groups; however, in the collagen group, it was scarce. At the middle timepoint, outgrowth from SCSCs was superior in both HA-based groups compared to collagen, regardless of the presence of NF. In DRGCs, the outgrowth in Healon 5® with NF was significantly higher compared to the rest of the groups. PCR revealed upregulation of NeuN gene expression in the HA-based groups compared to controls after excitotoxic injury. The differences in neurite outgrowth from the two different tissue models suggest that axons differentially respond to the two types of biomaterials.

Trabecular Titanium for Orthopedic Applications: Balancing Antimicrobial with Osteoconductive Properties by Varying Silver Contents.

Periprosthetic joint infection (PJI) and implant loosening are the most common complications after joint replacement surgery. Due to their increased surface area, additively manufactured porous metallic implants provide optimal osseointegration but they are also highly susceptible to bacterial colonization. Antibacterial surface coatings of porous metals that do not inhibit osseointegration are therefore highly desirable. The potential of silver coatings on arthroplasty implants to inhibit PJI has been demonstrated, but the optimal silver content and release kinetics have not yet been defined. A tight control over the silver deposition coatings can help overcome bacterial infections while reducing cytotoxicity to human cells. In this regard, porous titanium sputtered with silver and titanium nitride with increasing silver contents enabled controlling the antibacterial effect against common PJI pathogens while maintaining the metabolic activity of human primary cells. Electron beam melting additively manufactured titanium alloys, coated with increasing silver contents, were physico-chemically characterized and investigated for effects against common PJI pathogens. Silver contents from 7 at % to 18 at % of silver were effective in reducing bacterial growth and biofilm formation. Staphylococcus epidermidis was more susceptible to silver ions than Staphylococcus aureus. Importantly, all silver-coated titanium scaffolds supported primary human osteoblasts proliferation, differentiation, and mineralization up to 28 days. A slight reduction of cell metabolic activity was observed at earlier time points, but no detrimental effects were found at the end of the culture period. Silver release from the silver-coated scaffolds also had no measurable effects on primary osteoblast gene expression since similar expression of genes related to osteogenesis was observed regardless the presence of silver. The investigated silver-coated porous titanium scaffolds may thus enhance osseointegration while reducing the risk of biofilm formation by the most common clinically encountered pathogens.

3D-printed porous Ti6Al4V alloys with silver coating combine osteocompatibility and antimicrobial properties.

Additive manufacturing allows for the production of porous metallic implants for use in orthopaedics, providing excellent mechanical stability and osseointegration. However, the increased surface area of such porous implants also renders them susceptible to bacterial colonization. In this work, two trabecular porous Ti6Al4V alloys produced by electron beam melting were investigated for their osteocompatibility and antimicrobial effects, comparing samples with a silver-coated surface to uncoated samples. Dense grit-blasted Ti samples were used for comparison. The porous samples had pore sizes of 500-600 µm and 5 to 10 µm surface roughness, the silver-coated samples contained 7 at.% Ag, resulting in a cumulative Ag release of 3.5 ppm up to 28 days. Silver reduced the adhesion of Staphylococcus aureus to porous samples and inhibited 72 h biofilm formation by Staphylococcus epidermidis but not that of S. aureus. Primary human osteoblast adhesion, proliferation and differentiation were not impaired in the presence of silver, and expression of osteogenic genes as well as production of mineralized matrix were similar on silver-coated and uncoated samples. Our findings indicate that silver coating of porous titanium implants can achieve antimicrobial effects without compromising osteocompatibility, but higher silver contents may be needed to yield a sustained protection against fast-growing bacteria.

Hexagonal pore geometry and the presence of hydroxyapatite enhance deposition of mineralized bone matrix on additively manufactured polylactic acid scaffolds.

Additive manufacturing (AM) has revolutionized the design of regenerative scaffolds for orthopaedic applications, enabling customizable geometric designs and material compositions that mimic bone. However, the available evidence is contradictory with respect to which geometric designs and material compositions are optimal. There is a lack of studies that systematically compare different pore sizes and geometries in conjunction with the presence or absence of calcium phosphates. We therefore evaluated the physicochemical and biological properties of additively manufactured scaffolds based on polylactic acid (PLA) in combination with hydroxyapatite (HA). HA was either incorporated in the polymeric matrix or introduced as a coating, yielding 15 and 2% wt., respectively. Pore sizes of the scaffolds varied between 200 and 450 µm and were shaped either triangularly or hexagonally. All scaffolds supported the adhesion, proliferation and differentiation of both primary mouse osteoblasts and osteosarcoma cells up to four weeks, with only small differences in the production of alkaline phosphatase (ALP) between cells grown on different pore geometries and material compositions. However, mineralization of the PLA scaffolds was substantially enhanced in the presence of HA, either embedded in the PLA matrix or as a coating at the surface level, and by larger hexagonal pores. In conclusion, customized HA/PLA composite porous scaffolds intended for the repair of critical size bone defects were obtained by a cost-effective AM method. Our findings indicate that the analysis of osteoblast adhesion and differentiation on experimental scaffolds alone is inconclusive without the assessment of mineralization, and the effects of geometry and composition on bone matrix deposition must be carefully considered in order to understand the regenerative potential of experimental scaffolds.

Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury.

Secondary damage following spinal cord injury (SCI) induces neuronal damage through inflammatory and excitotoxic pathways. We hypothesized that the interleukin-1 receptor antagonist (IL1RA) protects neuronal populations and suppresses apoptosis and gliosis after injury. Spinal cord slice cultures (SCSCs) were subjected to excitotoxic injury with N-methyl-D-aspartate (NMDA) and treated with IL1RA. Immunohistochemistry for neuronal nuclei (NeuN), MacII, glial fibrillary acidic protein, and TdT-mediated dUTP nick end labelling stains were used to evaluate neuronal survival, glial activation, and apoptosis. Treatment with IL1RA significantly reduced the number of apoptotic cells in both NMDA-lesioned and unlesioned cultures. Experimental injury with NMDA reduced the number of NeuN-positive ventral horn neurons, and IL1RA treatment counteracted this loss 1 day after injury. However, IL1RA had no effect on the number of presumable Renshaw cells, identified by their selective expression of the cholinergic nicotinic α2-receptor subunit (Chrna2). Activated microglial cells were more numerous in NMDA-lesioned cultures 1 day after injury, and IL1RA significantly reduced their numbers. We conclude that IL1RA modulates neuronal apoptosis and microglial activation in excitotoxically injured SCSCs. Renshaw cells were more susceptible to excitotoxic injury than other neurons and were not rescued by IL1RA treatment. Modulation of IL-1-mediated pathways may thus be effective in reducing excitotoxically induced neuronal damage after SCI, however only in specific neuronal populations, such as ventral horn neurons. These findings motivate further investigations of the possibility to antagonize inflammatory pathways after SCI.

A uni-cortical femoral defect model in the rat: evaluation using injectable hyaluronan hydrogel as a carrier for bone morphogenetic protein-2.

The development of biomaterial for bone regeneration requires animal models that are reliable and designed to mimic clinically relevant situations. We have previously investigated hydrogels comprised of modified hyaluronic acid and polyvinyl alcohol in models of ectopic bone formation. This hydrogel induces bone regeneration when loaded with bone morphogenetic proteins (BMPs). To allow further optimization of hydrogels, we developed a new, femoral, non-critical-sized cortical defect model. In the rat femur, we drilled standardized, elongated unilateral cortical defects that did not require stabilization and that could be created bilaterally to allow paired comparisons of biomaterials. After optimizing the defect size, subsequent stress fractures occurred in only 8% and the defect healed partially over the 40 day study period. In a time-course experiment, we treated bone defects with the previously studied hyaluronan hydrogel loaded with 10 µg hydroxyapatite and 6 µg BMP-2. The shape of the defect allowed controlled containment of the material within the defect. The defect in the right leg was left untreated, while the left defect was filled with 40 µl of the BMP hydrogel. As determined by pQCT analysis, the treated defects had a higher bone mineral content, bone area and bone density than control defects. The relative difference was greatest between the groups at 10 and 20 days and diminished as the defect healed in the untreated legs. We conclude that this animal model allows facile and rapid screening of biomaterials for bone regeneration in cortical femoral defects without requiring external fixation.

Hyaluronic acid-based hydrogel enhances neuronal survival in spinal cord slice cultures from postnatal mice.

Numerous biomaterials based on extracellular matrix-components have been developed. It was our aim to investigate whether a hyaluronic acid-based hydrogel improves neuronal survival and tissue preservation in organotypic spinal cord slice cultures. Organotypic spinal cord slice cultures were cultured for 4 days in vitro (div), either on hyaluronic acid-based hydrogel (hyaluronic acid-gel group), collagen gel (collagen group), directly on polyethylene terephthalate membrane inserts (control group), or in the presence of soluble hyaluronic acid (soluble hyaluronic acid group). Cultures were immunohistochemically stained against neuronal antigen NeuN and analyzed by confocal laser scanning microscopy. Histochemistry for choline acetyltransferance, glial fibrillary acidic protein, and Griffonia simplicifolia isolectin B4 followed by quantitative analysis was performed to assess motorneurons and different glial populations. Confocal microscopic analysis showed a 4-fold increase in the number of NeuN-positive neurons in the hyaluronic acid-gel group compared to both collagen (p < 0.001) and control groups (p < 0.001). Compared to controls, organotypic spinal cord slice cultures maintained on hyaluronic acid-based hydrogel showed 5.9-fold increased survival of choline acetyltransferance-positive motorneurons (p = 0.008), 2-fold more numerous resting microglial cells in the white matter (p = 0.031), and a 61.4% reduction in the number of activated microglial cells within the grey matter (p = 0.05). Hyaluronic acid-based hydrogel had a shear modulus (G') of ≈1200 Pascals (Pa), which was considerably higher than the ≈25 Pa measured for collagen gel. Soluble hyaluronic acid failed to improve tissue preservation. In conclusion, hyaluronic acid-based hydrogel improves neuronal and - most notably - motorneuron survival in organotypic spinal cord slice cultures and microglial activation is limited. The positive effects of hyaluronic acid-based hydrogel may at least in part be due to its mechanical properties.

Morphological differences in BMP-2-induced ectopic bone between solid and crushed hyaluronan hydrogel templates.

The possibility to affect bone formation by using crushed versus solid hydrogels as carriers for bone morphogenetic protein 2 (BMP-2) was studied. Hydrogels, based on chemical crosslinking between hyaluronic acid and poly(vinyl alcohol) derivatives, were loaded with BMP-2 and hydroxyapatite. Crushed and solid forms of the gels were analyzed both in vitro via a release study using ¹²5I radioactive labeling of BMP-2, and in vivo in a subcutaneous ectopic bone model in rats. Dramatically different morphologies were observed for the ectopic bone formed in vivo in the two types of gels, even though virtually identical release profiles were observed in vitro. Solid hydrogels induced formation of a dense bone shell around non-degraded hydrogel, while crushed hydrogels demonstrated a uniform bone formation throughout the entire sample. These results suggest that by crushing the hydrogel, the construct's three-dimensional network becomes disrupted. This could expose unreacted functional groups, making the fragment's surfaces reactive and enable limited chemical fusion between the crushed hydrogel fragments, leading to similar in vitro release profiles. However, in vivo these interactions could be broken by enzymatic activity, creating a macroporous structure that allows easier cell infiltration, thus, facilitating bone formation.

3-Aryl pyridone derivatives. Potent and selective kappa opioid receptor agonists.

A new series of 3-aryl pyridone based kappa opioid receptor agonists was designed and synthesised, based on an understanding of the classical kappa opioid receptor pharmacophore. The most potent of the new compounds were comparable to U-69,593 in receptor affinity, selectivity and functional agonist effect at the cloned human kappa opioid receptor.