The low in vitro stability of dynamic covalent hydrogels with fast exchanging bonds prevents their long-term applications despite their benefits, like fast self-healing and stress relaxation. To circumvent this while maintaining the fast dynamics, Schiff base crosslinked alginate hydrogels were developed with different crosslinking chemistry and structure. Alginate dialdehyde (ADA) was synthesized and used to prepare hydrazone and oxime crosslinked hydrogels. Two crosslinking strategies were followed, one group was prepared by ADA and difunctional small molecule crosslinkers having hydrazide and oxyamine functionalities. The other group was prepared by hydrazide-modified alginate and ADA to study the effects of changing the crosslinker structure while maintaining the crosslinking chemistry on the hydrogel dynamics. The effects of the polymer concentration, crosslinking chemistry, and structure on the hydrogel properties, self-healing ability, and injectability were evaluated. Mitigating the low in vitro stability, the fast exchange of dynamic bonds was exploited to control the delivery of L-Arginine (L-Arg). The release of L-Arg was assessed at different pH to investigate the stimuli-responsiveness of the hydrogels and was compared with the release from ionically crosslinked hydrogels. Furthermore, the effects of incorporating L-Arg as a competitor in forming imine bonds on the mechanical properties of the hydrogels were investigated.

Stimuli-responsive polymeric nanoparticles (NPs) can serve as smart drug delivery systems (DDSs) by triggering drug release upon external or internal stimuli. A dual-responsive DDS made of a triblock poly(ϵ-caprolactone)-poly(ethylene glycol)-poly(ϵ-caprolactone) (PCL-SS-PEG-SS-PCL) copolymer, bearing disulfide bonds between PCL and PEG, was synthesized. The copolymer was functionalized with coumarin and sensitive to near-infrared (NIR) light irradiation, while the S-S bonds could be cleaved by GSH (10 mM). Characterization was achieved by nuclear magnetic resonance, size exclusion chromatography, and Fourier transform infrared analyses. Nile Red (NR)-loaded NPs were prepared through self-assembly of the copolymer in water and analyzed by dynamic light scattering and field-emission scanning electron microscopy. The NR release upon ultraviolet (UV)/NIR light irradiation as well as by GSH concentrations was monitored by using fluorescence spectroscopy, while simultaneous exposure to UV/NIR light and intracellular GSH concentration led to faster NR release. AlamarBlue assay showed satisfactory cell viability of the NR-loaded NPs, while their cellular uptake in human dermal fibroblast cells was investigated by fluorescence microscopy and fluorescence emission measurements.

In the field of tissue engineering, synthetic and degradable polyesters like poly(ε-caprolactone) (PCL) and poly(ε-caprolactone-co-p-dioxanone) (PCLDX) are widely used as scaffolds. Our previous research revealed that thermal storage conditions could alter the surface texture of PCL and PCLDX scaffolds, which might influence cell-scaffold interactions in tissue engineering applications. These findings highlighted the importance of multi-scale characterization techniques to identify the scales most sensitive to external changes and the need for personalized surface texture analysis. Sterilization techniques, such as ethylene oxide and gamma radiation, are essential for ensuring the sterility of polymeric medical devices. However, these processes can significantly impact the bulk polymer properties and/or surface texture of the scaffolds, potentially affecting their biocompatibility, safety, and overall performance. Therefore, the influence of sterilization processes on the surface texture of PCLDX films and electrospun nanofibers and to correlate these findings with the thermal and physical properties of the polymer are essential and have been assessed. Our results demonstrated that ethylene oxide maintained the structural integrity and surface texture of PCLDX scaffolds, while gamma irradiation caused a significant reduction in molar mass and increased the number of hills (Shn) and dales (Sdn) on PCLDX samples. Despite these changes, both sterilization methods showed minimal effects on the thermal properties, such as melting temperature and degree of crystallinity, and surface wettability of the scaffolds. This comprehensive surface texture analysis highlights the importance of evaluating feature parameters such as Shn and Sdn for optimizing the performance and biocompatibility of polymeric scaffolds in tissue engineering.

The fast-progressing landscape of the bioprocessing industry emphasizes innovation and efficiency enhancement, propelled by the integration of advanced solutions. Additive manufacturing technologies, particularly laserbased powder bed fusion with polypropylene, are pivotal in this industrial metamorphosis. However, despite the substantial scientific effort in the field, a significant gap exists in comprehending the surface characteristics of new surfaces and their implications for bacterial attachment and biofilm formation. This arises, in part, due to the absence of comprehensive and universally applicable topographical characterization analysis specifically designed for additively manufactured-fabricated surfaces. Typically, researchers tend to rely on the commonly used roughness parameter, Sa, that primarily quantifies the average height variation across a surface. Addressing this limitation is crucial for understanding the connection between surface characteristics and bacterial attachment dynamics. Here, we propose an innovative approach using surface analysis including confocal microscopy, advanced roughness measurements, and multivariate statistical analysis to uncover the connections between bacterial attachment for Gram negative Escherichia coli and Gram positive Staphylococcus aureus in early biofilm formation with surfaces produced by standardized and additively manufactured techniques. Finally, we advocate for the adoption of a set of roughness parameters that specifically describe the dale region of the surfaces. By doing so, we intend to establish direct links between surface texture and bacterial adhesion, thus contributing significantly to the advancement of both bioprocessing and additive manufacturing research domains.

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.

Light responsive polymeric materials are of great interest for several applications; in particular, the NIR spectrum is considered an ideal light source for e.g. biomedical applications. Here, we have designed light sensitive poly (epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL) triblock copolymer, end-capped with modified coumarin. When dialyzed against water, the coumarin-functionalized triblock copolymers self -assembled into core-shell nanoparticles, characterized by DLS, SEM, and NMR. The hydrophobic Nile Red, used as a model guest molecule, was loaded in the nanoparticles and its release by light irradiation of selected wavelength was demonstrated by fluorescence spectroscopy. When irradiated with UV or NIR light, coumarin end-groups of the copolymer were photolyzed, converting the end groups to carboxylic acids, thus provoking the disruption of the preassembled nanoparticles, and the release of the encapsulated guest molecule.

Background: The repair and treatment of large bone defects in patients with compromised bone metabolism due to ageing and medical conditions such as osteoporosis present often a clinical challenge. Therefore, adjunctive methods to enhance bone healing are needed.

Bone tissue engineering with application of nanotechnology allows to construct biomaterials with desired properties being osteoconductive, osteoinductive and osteogenic.

Aim / Hypothesis: The aim of our study was to promote bone regeneration using functionalised scaffold with Rhamnogalacturonan-I pectins (RG-I) in vitro and in vivo using aging and osteoporotic rodent models.

Material and Methods: The biomaterials were poly(l-lactide-co-ε-caprolactone) scaffolds and the RG-I was from potato. The chemical and physical properties of functionalised biomaterials with RG-I nanoparticles were characterised using confocal and atomic force microscopy. Functionalised scaffolds with RG-I (tested sample) were evaluated in vitro with human osteoblasts from osteoporotic patients and their response was tested using real-time PCR. In vivo evaluation was performed using critical-size calvaria bone defect model in ageing and osteoporotic rat models. Scaffolds were implanted randomly in the calvaria defects of aged female Wistar rats (11-12 months old) and osteoporotic female Wistar rats induced by ovariectomy. The control was scaffold without RG-I. After 2 and 8 weeks, animals were euthanised. Harvested samples were analysed for osteogenic and inflammatory markers using real-time PCR. Bone formation was evaluated radiographically and histologically. The data was analysed using one-way ANOVA.

Results: The chemical and physical properties results indicated success of the functionalisation of scaffolds with RG-I. Osteoblasts response suggested osteogenic (upregulation osteopontin, osteocalcin, collagen1, bone sialoprotein) and anti-inflammatory properties (downregulation IL-1, IL-8, TNF-alpha) on the scaffold functionalised with RG-I. The in vivo results in aged and osteoporotic rat calvaria model of early (2 weeks) bone regeneration showed increase of osteogenic markers and decrease of proinflammatory markers and RANKL, compared to control. In osteoporotic rat model at week 2 and 8 and in aged rat model at week 8, the mean percentage of BV / TV (bone volume / tissue volume) in the defect with RG-I scaffold was significantly greater than the defect with control. The histological evaluation in both rat models revealed larger areas of new bone formation in RG-I scaffolds than in control.

Conclusion and Clinical implications: In conclusion, the plant-derived nanoparticles significantly increased osteogenic and decreased pro-inflammatory response in vitro and in vivo. These finding may have a crucial impact on bone repair process especially in elderly and osteoporotic patients.

Fused filament fabrication (FFF) is a commonly used method for producing three-dimensional scaffolds using synthetic, degradable polymers. However, there are several variables that must be considered when fabricating devices for clinical use, one of which is storage conditions after printing. While the academic community has examined the impact of FFF on mechanical and thermal properties, there has been less focus on how storage conditions would affect the surface texture of scaffolds. Our hypothesis was that the surface, thermal and physical properties of FFF scaffolds are significantly influenced by the storage conditions. We evaluated the surfaces of FFF poly (ε-caprolactone) (PCL) and poly (ε-caprolactone-co-p-dioxanone) (PCLDX) strands that were stored at 4 °C, 20 °C, and 37 °C for 28 days. We monitored surface texture, physical and thermal changes to understand the effect of storage on the strands. The implementation of scale-sensitive fractal analysis and feature parameters revealed that storage conditions at 37 °C increased the number of hills and dales, as well as the density of peaks and pits compared to 20 °C and 4 °C, for both materials. The feature roughness parameters for PCL had up to 90% higher values than those of PCLDX, which correlated with the physical and thermal properties of the materials. These differences may impact further surface-cell interaction, highlighting the need for further evaluation for faster clinical translation. Our findings emphasize the importance of considering storage conditions in the design and manufacture of FFF scaffolds and suggest that the use of feature roughness parameters could facilitate the optimization and tailoring the surface properties for specific applications.