Electrophoretically Co-Deposited Collagen-Lactoferrin Membranes with Enhanced Pro-Regenerative Properties for Oral Soft Tissue Regeneration.

The quality of soft tissue defect regeneration after dental surgeries largely determines their final success. Collagen membranes have been proposed for the healing of such defects, but in some cases, they do not guarantee a sufficient volume of the regenerated tissue and vascularization. For this purpose, lactoferrin, a protein with natural pro-regenerative, anti-inflammatory, and pro-angiogenic activity, can be added to collagen. In this article, we used a semipermeable barrier-assisted electrophoretic deposition (SBA-EPD) method for the production of collagen-lactoferrin membranes. The membrane structure was studied by SEM, and its mechanical properties were shown. The lactoferrin release kinetics were shown by ELISA within 75 h. When tested in vitro, we demonstrated that the collagen-lactoferrin membranes significantly increased the proliferation of keratinocytes (HaCaT) and fibroblasts (977hTERT) compared to blank collagen membranes. In vivo, on the vestibuloplasty and free gingival graft harvesting models, we showed that collagen-lactoferrin membranes decreased the wound inflammation and increased the healing rates and regeneration quality. In some parameters, collagen-lactoferrin membranes outperformed not only blank collagen membranes, but also the commercial membrane Mucograft®. Thus, we proved that collagen-lactoferrin membranes produced by the SBA-EPD method may be a valuable alternative to commercially used membranes for soft tissue regeneration in the oral cavity.

3D or not 3D: a guide to assess cell viability in 3D cell systems.

Cell viability is the primary integrative parameter used for various purposes, particularly when fabricating tissue equivalents (e.g., using bioprinting or scaffolding techniques), optimizing conditions to cultivate cells, testing chemicals, drugs, and biomaterials, etc. Most of the conventional methods were originally designed for a monolayer (2D) culture; however, 2D approaches fail to adequately assess a tissue-engineered construct's viability and drug effects and recapitulate the host-pathogen interactions and infectivity. This study aims at revealing the influence of particular 3D cell systems' parameters such as the components' concentration, gel thickness, cell density, etc. on the cell viability and applicability of standard assays. Here, we present an approach to achieving adequate and reproducible results on the cell viability in 3D collagen- and fibrin-based systems using the Live/Dead, AlamarBlue, and PicoGreen assays. Our results have demonstrated that a routine precise analysis of 3D systems should be performed using a combination of at least three methods based on different cell properties, e.g. the metabolic activity, proliferative capacity, morphology, etc.

Lactoferrin as a regenerative agent: The old-new panacea?

Lactoferrin (Lf) possesses various biological properties and therapeutic potentials being a perspective anti-inflammatory, antibacterial, antiviral, antioxidant, antitumor, and immunomodulatory agent. A significant body of literature has also demonstrated that Lf modulates regenerative processes in different anatomical structures, such as bone, cartilage, skin, mucosa, cornea, tendon, vasculature, and adipose tissue. Hence, this review collected and analyzed the data on the regenerative effects of Lf, as well as paid specific attention to their molecular basis. Furthermore, tissue and condition-specific activities of different Lf types as well as problems of their delivery to the targeted organs were discussed. The authors strongly hope that this review will stimulate researchers to focus on the highlighted topics thus accelerating the progress of Lf's wider clinical application.

From Aggregates to Porous Three-Dimensional Scaffolds through a Mechanochemical Approach to Design Photosensitive Chitosan Derivatives.

The crustacean processing industry produces large quantities of waste by-products (up to 70%). Such wastes could be used as raw materials for producing chitosan, a polysaccharide with a unique set of biochemical properties. However, the preparation methods and the long-term stability of chitosan-based products limit their application in biomedicine. In this study, different scale structures, such as aggregates, photo-crosslinked films, and 3D scaffolds based on mechanochemically-modified chitosan derivatives, were successfully formed. Dynamic light scattering revealed that aggregation of chitosan derivatives becomes more pronounced with an increase in the number of hydrophobic substituents. Although the results of the mechanical testing revealed that the plasticity of photo-crosslinked films was 5⁻8% higher than that for the initial chitosan films, their tensile strength remained unchanged. Different types of polymer scaffolds, such as flexible and porous ones, were developed by laser stereolithography. In vivo studies of the formed structures showed no dystrophic and necrobiotic changes, which proves their biocompatibility. Moreover, the wavelet analysis was used to show that the areas of chitosan film degradation were periodic. Comparing the results of the wavelet analysis and X-ray diffraction data, we have concluded that degradation occurs within less ordered amorphous regions in the polymer bulk.

Collagen Matrix to Restore the Tympanic Membrane: Developing a Novel Platform to Treat Perforations.

Modern otology faces challenges in treating tympanic membrane (TM) perforations. Instead of surgical intervention, alternative treatments using biomaterials are emerging. Recently, we developed a robust collagen membrane using semipermeable barrier-assisted electrophoretic deposition (SBA-EPD). In this study, a collagen graft shaped like a sponge through SBA-EPD was used to treat acute and chronic TM perforations in a chinchilla model. A total of 24 ears from 12 adult male chinchillas were used in the study. They were organized into four groups. The first two groups had acute TM perforations and the last two had chronic TM perforations. We used the first and third groups as controls, meaning they did not receive the implant treatment. The second and fourth groups, however, were treated with the collagen graft implant. Otoscopic assessments were conducted on days 14 and 35, with histological evaluations and TM vibrational studies performed on day 35. The groups treated with the collagen graft showed fewer inflammatory changes, improved structural recovery, and nearly normal TM vibrational properties compared to the controls. The porous collagen scaffold successfully enhanced TM regeneration, showing high biocompatibility and biodegradation potential. These findings could pave the way for clinical trials and present a new approach for treating TM perforations.

Oral Galvanism Side Effects: Comparing Alloy Ions and Galvanic Current Effects on the Mucosa-like Model.

The interaction of different dental alloys with the oral environment may cause severe side effects (e.g., burning sensation, inflammatory reactions, carcinogenesis) as a result of oral galvanism. However, the pathogenesis of side effects associated with oral galvanism is still unclear, and the effects of direct current and alloy corrosion ions are considered potentially contributing factors. Therefore, the aim of this study was to systemically compare the damaging effects of (1) galvanism as a synergistic process (direct current + corrosion ions), (2) direct current separately, and (3) corrosion ions separately on an in vitro mucosa-like model based on a cell line of immortalized human keratinocytes (HaCaTs) to reveal the factors playing a pivotal role in dental alloys side effects. For this, we chose and compared the dental alloys with the highest risk of oral galvanism: Ti64-AgPd and NiCr-AgPd. We showed that galvanic current may be the leading damaging factor in the cytotoxic processes associated with galvanic coupling of metallic intraoral appliances in the oral cavity, especially in the short-term period (28 days). However, the contribution of corrosion ions (Ni2+) to the synergistic toxicity was also shown, and quite possibly, in the long term, it could be no less dangerous.

The Impact of Commercially Available Dry Mouth Products on the Corrosion Resistance of Common Dental Alloys.

Dental implants are thought to be implanted for life, but throughout their lifespan, they function in aggressive oral environment, resulting in corrosion of the material itself as well as possible inflammation of adjacent tissues. Therefore, materials and oral products for people with metallic intraoral appliances must be chosen carefully. The purpose of this study was to investigate the corrosion behavior of common titanium and cobalt-chromium alloys in interaction with various dry mouth products using electrochemical impedance spectroscopy (EIS). The study showed that different dry mouth products lead to different open circuit potentials, corrosion voltages, and currents. The corrosion potentials of Ti64 and CoCr ranged from -0.3 to 0 V and -0.67 to 0.7 V, respectively. In contrast to titanium, pitting corrosion was observed for the cobalt-chromium alloy, leading to the release of Co and Cr ions. Based on the results, it can be argued that the commercially available dry mouth remedies are more favorable for dental alloys in terms of corrosion compared to Fusayama Meyer's artificial saliva. Thus, to prevent undesirable interactions, the individual characteristics of not only the composition of each patient's tooth and jaw structure, but also the materials already used in their oral cavity and oral hygiene products, must be taken into account.

Laser Bioprinting with Cell Spheroids: Accurate and Gentle.

Laser printing with cell spheroids can become a promising approach in tissue engineering and regenerative medicine. However, the use of standard laser bioprinters for this purpose is not optimal as they are optimized for transferring smaller objects, such as cells and microorganisms. The use of standard laser systems and protocols for the transfer of cell spheroids leads either to their destruction or to a significant deterioration in the quality of bioprinting. The possibilities of cell spheroids printing by laser-induced forward transfer in a gentle mode, which ensures good cell survival ~80% without damage and burns, were demonstrated. The proposed method showed a high spatial resolution of laser printing of cell spheroid geometric structures at the level of 62 ± 33 µm, which is significantly less than the size of the cell spheroid itself. The experiments were performed on a laboratory laser bioprinter with a sterile zone, which was supplemented with a new optical part based on the Pi-Shaper element, which allows for forming laser spots with different non-Gaussian intensity distributions. It is shown that laser spots with an intensity distribution profile of the "Two rings" type (close to Π-shaped) and a size comparable to a spheroid are optimal. To select the operating parameters of laser exposure, spheroid phantoms made of a photocurable resin and spheroids made from human umbilical cord mesenchymal stromal cells were used.

Minimization of Adverse Effects Associated with Dental Alloys.

Metal alloys are one of the most popular materials used in current dental practice. In the oral cavity, metal structures are exposed to various mechanical and chemical factors. Consequently, metal ions are released into the oral fluid, which may negatively affect the surrounding tissues and even internal organs. Adverse effects associated with metallic oral appliances may have various local and systemic manifestations, such as mouth burning, potentially malignant oral lesions, and local or systemic hypersensitivity. However, clear diagnostic criteria and treatment guidelines for adverse effects associated with dental alloys have not been developed yet. The present comprehensive literature review aims (1) to summarize the current information related to possible side effects of metallic oral appliances; (2) to analyze the risk factors aggravating the negative effects of dental alloys; and (3) to develop recommendations for diagnosis, management, and prevention of pathological conditions associated with metallic oral appliances.

Laser-induced Forward Transfer Hydrogel Printing: A Defined Route for Highly Controlled Process.

Laser-induced forward transfer is a versatile, non-contact, and nozzle-free printing technique which has demonstrated high potential for different printing applications with high resolution. In this article, three most widely used hydrogels in bioprinting (2% hyaluronic acid sodium salt, 1% methylcellulose, and 1% sodium alginate) were used to study laser printing processes. For this purpose, the authors applied a laser system based on a pulsed infrared laser (1064 nm wavelength, 8 ns pulse duration, 1 - 5 J/cm2 laser fluence, and 30 µm laser spot size). A high-speed shooting showed that the increase in fluence caused a sequential change in the transfer regimes: No transfer regime, optimal jetting regime with a single droplet transfer, high speed regime, turbulent regime, and plume regime. It was demonstrated that in the optimal jetting regime, which led to printing with single droplets, the size and volume of droplets transferred to the acceptor slide increased almost linearly with the increase of laser fluence. It was also shown that the maintenance of a stable temperature (±2°C) allowed for neglecting the temperature-induced viscosity change of hydrogels. It was determined that under room conditions (20°C, humidity 50%), the hydrogel layer, due to drying processes, decreased with a speed of about 8 µm/min, which could lead to a temporal variation of the transfer process parameters. The authors developed a practical algorithm that allowed quick configuration of the laser printing process on an applied experimental setup. The configuration is provided by the change of the easily tunable parameters: Laser pulse energy, laser spot size, the distance between the donor ribbon and acceptor plate, as well as the thickness of the hydrogel layer on the donor ribbon slide.

Coupling PEG-LZM polymer networks with polyphenols yields suturable biohydrogels for tissue patching.

Poor mechanical performances severely limit the application of hydrogels in vivo; for example, it is difficult to perform a very common suturing operation on hydrogels during surgery. There is a growing demand to improve the mechanical properties of hydrogels for broadening their clinical applications. Natural polyphenols can match the potential toughening sites in our previously reported PEG-lysozyme (LZM) hydrogel because polyphenols have unique structural units including a hydroxyl group and an aromatic ring that can interact with PEG via hydrogen bonding and form hydrophobic interactions with LZM. By utilizing polyphenols as noncovalent crosslinkers, the resultant PEG-LZM-polyphenol hydrogel presents super toughness and high elasticity in comparison to pristine PEG-LZM with no obvious changes in the initial shape, and it can even withstand the high pressure from sutures. At the same time, the mechanical properties could be widely adjusted by varying the polyphenol concentration. Interestingly, the PEG-LZM-polyphenol hydrogel has a higher water content than other polyphenol-toughened hydrogels, which may better meet the clinical needs for hydrogel materials. Besides, the introduction of polyphenols endows the hydrogel with improved antibacterial and anti-inflammatory abilities. Finally, the PEG-LZM-polyphenol (tannic acid) hydrogel was demonstrated to successfully patch a rabbit myocardial defect by suturing for 4 weeks and improve the wound healing and heart function recovery compared to autologous muscle patches.

Redox-Channeling Polydopamine-Ferrocene (PDA-Fc) Coating To Confer Context-Dependent and Photothermal Antimicrobial Activities.

Microbial disinfection associated with medical device surfaces has been an increasing need, and surface modification strategies such as antibacterial coatings have gained great interest. Here, we report the development of polydopamine-ferrocene (PDA-Fc)-functionalized TiO2 nanorods (Ti-Nd-PDA-Fc) as a context-dependent antibacterial system on implant to combat bacterial infection and hinder biofilm formation. In this work, two synergistic antimicrobial mechanisms of the PDA-Fc coating are proposed. First, the PDA-Fc coating is redox-active and can be locally activated to release antibacterial reactive oxygen species (ROS), especially ·OH in response to the acidic microenvironment induced by bacteria colonization and host immune responses. The results demonstrate that redox-based antimicrobial activity of Ti-Nd-PDA-Fc offers antibacterial efficacy of over 95 and 92% against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. Second, the photothermal effect of PDA can enhance the antibacterial capability upon near-infrared (NIR) irradiation, with over 99% killing efficacy against MRSA and E. coli, and even suppress the formation of biofilm through both localized hyperthermia and enhanced ·OH generation. Additionally, Ti-Nd-PDA-Fc is biocompatible when tested with model pre-osteoblast MC-3T3 E1 cells and promotes cell adhesion and spreading presumably due to its nanotopographical features. The MRSA-infected wound model also indicates that Ti-Nd-PDA-Fc with NIR irradiation can effectively eliminate bacterial infection and suppress host inflammatory responses. We believe that this study demonstrates a simple means to create biocompatible redox-active coatings that confer context-dependent antibacterial activities to implant surfaces.