Polycaprolactone/ß-Tricalcium Phosphate Composite Scaffolds with Advanced Pore Geometries Promote Human Mesenchymal Stromal Cells' Osteogenic Differentiation.

Critical-sized mandibular bone defects, arising from, for example, resections after tumor surgeries, are currently treated with autogenous bone grafts. This treatment is considered very invasive and is associated with limitations such as morbidity and graft resorption. Tissue engineering approaches propose to use 3D scaffolds that combine structural features, biomaterial properties, cells, and biomolecules to create biomimetic constructs. However, mimicking the complex anatomy and composition of the mandible poses a challenge in scaffold design. In our study, we evaluated the dual effect of complex pore geometry and material composition on the osteogenic potential of 3D printed scaffolds. The scaffolds were made of polycaprolactone (PCL) alone (TCP0), or with a high concentration of ß-tricalcium phosphate (ß-TCP) up to 40% w/w (TCP40), with two complex pore geometries, namely a star- (S) and a diamond-like (D) shape. Scanning electron microscopy and microcomputed tomography images confirmed high fidelity during the printing process. The D-scaffolds displayed higher compressive moduli than the corresponding S-scaffolds. TCP40 scaffolds in simulated body fluid showed deposition of minerals on the surface after 28 days. Subsequently, we assessed the differentiation of seeded bone marrow-derived human mesenchymal stromal cells (hMSCs) over 28 days. The early expression of RUNX2 in the cell nuclei confirmed the commitment toward an osteogenic phenotype. Moreover, alkaline phosphatase (ALP) activity and collagen deposition displayed an increasing trend in the D-scaffolds. Collagen type I was mainly present in the deposited extracellular matrix (ECM), confirming deposition of bone matrix. Finally, Alizarin Red staining showed successful mineralization on all the TCP40 samples, with higher values for the S-shaped scaffolds. Taken together, our study demonstrated that the complex pore architectures of scaffolds comprised TCP40 stimulated osteogenic differentiation and mineralization of hMSCs in vitro. Future research will aim to validate these findings in vivo.

Microscopic and Macroscopic Characterization of Hydrogels Based on Poly(vinyl-alcohol)-Glutaraldehyde Mixtures for Fricke Gel Dosimetry.

In recent decades, hydrogels have emerged as innovative soft materials with widespread applications in the medical and biomedical fields, including drug delivery, tissue engineering, and gel dosimetry. In this work, a comprehensive study of the macroscopic and microscopic properties of hydrogel matrices based on Poly(vinyl-alcohol) (PVA) chemically crosslinked with Glutaraldehyde (GTA) was reported. Five different kinds of PVAs differing in molecular weight and degree of hydrolysis were considered. The local microscopic organization of the hydrogels was studied through the use of the 1H nuclear magnetic resonance relaxometry technique. Various macroscopic properties (gel fraction, water loss, contact angle, swelling degree, viscosity, and Young's Modulus) were investigated with the aim of finding a correlation between them and the features of the hydrogel matrix. Additionally, an optical characterization was performed on all the hydrogels loaded with Fricke solution to assess their dosimetric behavior. The results obtained indicate that the degree of PVA hydrolysis is a crucial parameter influencing the structure of the hydrogel matrix. This factor should be considered for ensuring stability over time, a vital property in the context of potential biomedical applications where hydrogels act as radiological tissue-equivalent materials.

MosChito rafts as a promising biocontrol tool against larvae of the common house mosquito, Culex pipiens.

Mosquito control is of paramount importance, in particular, in light of the major environmental alterations associated with human activities, from climate change to the altered distribution of pathogens, including those transmitted by Arthropods. Here, we used the common house mosquito, Culex pipiens to test the efficacy of MosChito raft, a novel tool for mosquito larval control. MosChito raft is a floating hydrogel matrix, composed of chitosan, genipin and yeast cells, as bio-attractants, developed for the delivery of a Bacillus thuringiensis israeliensis (Bti)-based bioinsecticide to mosquito larvae. To this aim, larvae of Cx. pipiens were collected in field in Northern Italy and a novel colony of mosquito species (hereafter: Trescore strain) was established. MosChito rafts, containing the Bti-based formulation, were tested on Cx. pipiens larvae from the Trescore strain to determine the doses to be used in successive experiments. Thus, bioassays with MosChito rafts were carried out under semi-field conditions, both on larvae from the Trescore strain and on pools of larvae collected from the field, at different developmental stages. Our results showed that MosChito raft is effective against Cx. pipiens. In particular, the observed mortality was over 50% after two days exposure of the larvae to MosChito rafts, and over 70-80% at days three to four, in both laboratory and wild larvae. In conclusion, our results point to the MosChito raft as a promising tool for the eco-friendly control of a mosquito species that is not only a nuisance insect but is also an important vector of diseases affecting humans and animals.

Soft Perfusable Device to Culture Skeletal Muscle 3D Constructs in Air.

Devices for in vitro culture of three-dimensional (3D) skeletal muscle tissues have multiple applications, including tissue engineering and muscle-powered biorobotics. In both cases, it is crucial to recreate a biomimetic environment by using tailored scaffolds at multiple length scales and to administer prodifferentiative biophysical stimuli (e.g., mechanical loading). On the contrary, there is an increasing need to develop flexible biohybrid robotic devices capable of maintaining their functionality beyond laboratory settings. In this study, we describe a stretchable and perfusable device to sustain cell culture and maintenance in a 3D scaffold. The device mimics the structure of a muscle connected to two tendons: Tendon-Muscle-Tendon (TMT). The TMT device is composed of a soft (E ∼ 6 kPa) porous (pore diameter: ∼650 µm) polyurethane scaffold, encased within a compliant silicone membrane to prevent medium evaporation. Two tendon-like hollow channels interface the scaffold with a fluidic circuit and a stretching device. We report an optimized protocol to sustain C2C12 adhesion by coating the scaffold with polydopamine and fibronectin. Then, we show the procedure for the soft scaffold inclusion in the TMT device, demonstrating the device's ability to bear multiple cycles of elongations, simulating a protocol for cell mechanical stimulation. By using computational fluid dynamic simulations, we show that a flow rate of 0.62 mL/min ensures a wall shear stress value safe for cells (<2 Pa) and 50% of scaffold coverage by an optimal fluid velocity. Finally, we demonstrate the effectiveness of the TMT device to sustain cell viability under perfusion for 24 h outside of the CO2 incubator. We believe that the proposed TMT device can be considered an interesting platform to combine several biophysical stimuli, aimed at boosting skeletal muscle tissue differentiation in vitro, opening chances for the development of muscle-powered biohybrid soft robots with long-term operability in real-world environments.

Regeneration of Critical-Sized Mandibular Defects Using 3D-Printed Composite Scaffolds: A Quantitative Evaluation of New Bone Formation in In Vivo Studies.

Mandibular tissue engineering aims to develop synthetic substitutes for the regeneration of critical size defects (CSD) caused by a variety of events, including tumor surgery and post-traumatic resections. Currently, the gold standard clinical treatment of mandibular resections (i.e., autologous fibular flap) has many drawbacks, driving research efforts toward scaffold design and fabrication by additive manufacturing (AM) techniques. Once implanted, the scaffold acts as a support for native tissue and facilitates processes that contribute to its regeneration, such as cells infiltration, matrix deposition and angiogenesis. However, to fulfil these functions, scaffolds must provide bioactivity by mimicking natural properties of the mandible in terms of structure, composition and mechanical behavior. This review aims to present the state of the art of scaffolds made with AM techniques that are specifically employed in mandibular tissue engineering applications. Biomaterials chemical composition and scaffold structural properties are deeply discussed, along with strategies to promote osteogenesis (i.e., delivery of biomolecules, incorporation of stem cells, and approaches to induce vascularization in the constructs). Finally, a comparison of in vivo studies is made by taking into consideration the amount of new bone formation (NB), the CSD dimensions, and the animal model.

MosChito rafts as effective and eco-friendly tool for the delivery of a Bacillus thuringiensis-based insecticide to Aedes albopictus larvae.

Adult mosquito females, through their bites, are responsible for the transmission of different zoonotic pathogens. Although adult control represents a pillar for the prevention of disease spread, larval control is also crucial. Herein we characterized the effectiveness of a suitable tool, named "MosChito raft", for the aquatic delivery of a Bacillus thuringiensis var. israelensis (Bti) formulate, a bioinsecticide active by ingestion against mosquito larvae. MosChito raft is a floating tool composed by chitosan cross-linked with genipin in which a Bti-based formulate and an attractant have been included. MosChito rafts (i) resulted attractive for the larvae of the Asian tiger mosquito Aedes albopictus, (ii) induced larval mortality within a few hours of exposure and, more importantly, (iii) protected the Bti-based formulate, whose insecticidal activity was maintained for more than one month in comparison to the few days residual activity of the commercial product. The delivery method was effective in both laboratory and semi-field conditions, demonstrating that MosChito rafts may represent an original, eco-based and user-friendly solution for larval control in domestic and peri-domestic aquatic habitats such as saucers and artificial containers in residential or urban environments.

Arduino Automated Microwave Oven for Tissue Decalcification.

Decalcification of hard tissues such as bone and teeth is a complex process that requires using chemicals such as acids and chelating agents. Acids act faster than chelating agents, but they have a greater risk of damaging biological samples. Increasing the reaction speed of the chelating agent may solve this issue. There are several strategies to speed up this process, and using microwaves seems to be one of the most effective. However, lab-dedicated microwave ovens are expensive, and their purchase may seem unjustified. Therefore, a low-cost modification of a commercial microwave oven, consisting of an Arduino automation device, has been developed. The setup has proven reliable for continuous work, thanks to implementing an electronic safety circuit. In addition, it may reduce the decalcification time using a chelating agent, achieving optimal results regarding tissue preservation and quality of histological sections.

A simplified method for detecting Gram-positive and Gram-negative bacteria in dental histological samples: A preliminary and comparative study.

OBJECTIVE: Bacteria identification inside the dental tissue is a complex procedure requiring specific protocols. This study aimed to compare two classical Gram staining methods with a new staining method proposed by the authors to detect Gram-positive and Gram-negative bacteria in dental histological samples of human dentin. METHODS: Ten human teeth, extracted because of various pathologies, were decalcified, dehydrated, and paraffin-embedded. Then, approximately 100 serial sections of 4 µm thickness were made per sample. The serial sections were placed on glass slides and were stained according to Brown-Brenn, Brown-Hopps, and a proposed modification of Brown-Brenn staining. Both ATCC strains, smeared on glass slides, were stained following each method's instructions used in histological samples. RESULTS: From a qualitative evaluation, the Brown-Brenn method resulted in better staining of Gram-positive bacteria, while the authors' proposed staining technique was more oriented towards Gram-negative bacteria. On the other hand, the Brown-Hopps showed quite a balance in detecting Gram-positive and Gram-negative bacteria. Unlike the Brown-Brenn stain, the other two protocols showed better stainability of Gram-negative microorganisms in bacterial-smeared samples. CONCLUSION: All staining techniques evaluated in this article can identify bacteria, but the outcome can change according to the staining procedure used.

Poloxamer-Based Hydrogel as Drug Delivery System: How Polymeric Excipients Influence the Chemical-Physical Properties.

Thermogelling amphiphilic block copolymers have been widely investigated in the development of pharmaceutical drug carriers. In particular, thermosensitive gels based on poloxamer 407 (P407) have great potential for periodontal disease treatment, thanks to their ability to be liquid at room temperature and become viscous gels at body temperature. However, some problems, related to short in situ residence time, reduce their feasible clinical use. Thus, in order to improve the effective applicability of these materials, we studied how P407 thermogels are affected by the pH and by the inclusion of different hydrophilic polymers, used as excipients for increasing the gel stiffness. For this scope, a complete chemical-physical characterization of the synthesized gels is provided, in terms of determination of sol-gel transition temperature, viscosity and erosion degree. The data are correlated according to a statistical multivariate approach based on Principal Component Analysis and their mucoadhesion properties are also tested by Tapping mode-Atomic Force Microscopy (TM-AFM) imaging. Finally, we studied how the different P407 formulations are able to influence the release pathway of two antibacterial drugs (i.e., chlorhexidine digluconate and doxycycline hyclate) largely used in oral diseases.

Biodegradable floating hydrogel baits as larvicide delivery systems against mosquitoes.

Biological methods for mosquito larvae control are completely biodegradable and have null or limited effects on nontarget organisms. However, commercially available products have a low residual activity, with the consequent need for multiple applications that inevitably increase costs and the risk of resistance phenomena insurgence. Smart delivery systems made of hydrogels proved their efficacy in increasing the action duration of biolarvicides up to several months, but the lack of an efficient baiting mechanism to strongly attract the target pest remains a problem in practical applications. In this work, we investigated two novel hydrogel-based formulations of completely natural composition for baiting and killing larvae of Aedes albopictus mosquitos. The proposed materials consist of charged crosslinked polysaccharides (chitosan and cellulose) and are specifically manufactured to float in water, simulating organic matter usually present at breeding sites. Within the hydrogels' matrix, yeast colonies of Saccharomyces cerevisiae were embedded as phagostimulants alongside a biolarvicide (Bacillus thuringiensis var. israelensis (Bti)). Despite the similar chemical nature and structure, chitosan-based hydrogels exhibited a markedly superior baiting potential compared to those made of cellulose and also succeeded in efficiently killing mosquito larvae just after a few hours from administration. We are confident that the proposed smart delivery hydrogel made of chitosan can be an enabling tool to attract mosquito larvae towards biopesticides of different nature without delocalizing active ingredients away from the breeding site and to simultaneously increase their residual activity, thus holding the potential of minimizing environmental pollution related to pest control and vector-borne disease prevention.

Monolithic Three-Dimensional Functionally Graded Hydrogels for Bioinspired Soft Robots Fabrication.

Bioinspired soft robotics aims at reproducing the complex hierarchy and architecture of biological tissues within artificial systems to achieve the typical motility and adaptability of living organisms. The development of suitable fabrication approaches to produce monolithic bodies provided with embedded variable morphological and mechanical properties, typically encountered in nature, is still a technological challenge. Here we report on a novel manufacturing approach to produce three-dimensional functionally graded hydrogels (3D-FGHs) provided with a controlled porosity gradient conferring them variable stiffness. 3D-FGHs are fabricated by means of a custom-designed liquid foam templating (LFT) technique, which relies on the inclusion of air bubbles generated by a blowing agent into the monomer-based template solution during ultraviolet-induced photopolymerization. The 3D-FGHs' apparent Young's modulus ranges from 0.37 MPa (bulky hydrogel region) to 0.09 MPa (highest porosity region). A fish-shaped soft swimmer is fabricated to demonstrate the feasibility of the LFT technique to produce bioinspired robots. Mobility tests show a significant improvement in terms of swimming speed when the robot is provided with a graded body. The proposed manufacturing approach constitutes an enabling solution for the development of macroscopic functionally graded hydrogel-based structures usable in biomimetic underwater soft robotics applications.

Effects of the 3D Geometry Reconstruction on the Estimation of 3D Porous Scaffold Permeability.

3D scaffolds for tissue engineering typically need to adopt a dynamic culture to foster cell distribution and survival throughout the scaffold. It is, therefore, crucial to know fluids' behavior inside the scaffold architecture, especially for complex porous ones. Here we report a comparison between simulated and measured permeability of a porous 3D scaffold, focusing on different modeling parameters. The scaffold features were extracted by microcomputed tomography (µCT) and representative volume elements were used for the computational fluid-dynamic analyses. The objective was to investigate the sensitivity of the model to the degree of detail of the µCT image and the elements of the mesh. These findings highlight the pros and cons of the modeling strategy adopted and the importance of such parameters in analyzing fluid behavior in 3D scaffolds.

Combined Effects of Electrical Stimulation and Protein Coatings on Myotube Formation in a Soft Porous Scaffold.

Compared to two-dimensional cell cultures, three-dimensional ones potentially allow recreating natural tissue environments with higher accuracy. The three-dimensional approach is being investigated in the field of tissue engineering targeting the reconstruction of various tissues, among which skeletal muscle. Skeletal muscle is an electroactive tissue which strongly relies upon interactions with the extracellular matrix for internal organization and mechanical function. Studying the optimization of myogenesis in vitro implies focusing on appropriate biomimetic stimuli, as biochemical and electrical ones. Here we present a three-dimensional polyurethane-based soft porous scaffold (porosity ~ 86%) with a Young's modulus in wet conditions close to the one of natural skeletal muscle tissue (~ 9 kPa). To study the effect of external stimuli on muscle cells, we functionalized the scaffold with extracellular matrix components (laminin and fibronectin) and observed an increase in myoblast proliferation over three days. Furthermore, the combination between laminin coating and electrical stimulation resulted in more spread and thicker myotubes compared to non-stimulated samples and samples receiving the single (non-combined) inputs. These results pave the way to the development of mature muscle tissue within three-dimensional soft scaffolds, through the combination of biochemical and electrical stimuli.