Corrigendum to "Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus".

[This corrects the article DOI: 10.1016/j.heliyon.2023.e23107.].

Three-dimensional (3D) polylactic acid gradient scaffold to study the behavior of osteosarcoma cells under dynamic conditions.

This study adopts an in vitro method to recapitulate the behavior of Saos-2 cells, using a system composed of a perfusion bioreactor and poly-L-lactic acid (PLLA) scaffold fabricated using the low-cost thermally-induced phase separation (TIPS) technique. Four distinct scaffold morphologies with different pore sizes were fabricated, characterized by Scanning electron microscopy and micro-CT analysis and tested with osteosarcoma cells under static and dynamic environments to identify the best morphology for cellular growth. In order to accomplish this purpose, cell growth and matrix deposition of the Saos-2 osteosarcoma cell line were assessed using Picogreen and OsteoImage assays. The obtained data allowed us to identify the morphology that better promotes Saos-2 cellular activity in static and dynamic conditions. These findings provided valuable insights into scaffold design and fabrication strategies, emphasizing the importance of the dynamic culture to recreate an appropriate 3D osteosarcoma model. Remarkably, the gradient scaffold exhibits promise for osteosarcoma applications, offering the potential for targeted tissue engineering approaches.

Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus.

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created.

Different responses of mice and rats hippocampus CA1 pyramidal neurons to in vitro and in vivo-like inputs.

The fundamental role of any neuron within a network is to transform complex spatiotemporal synaptic input patterns into individual output spikes. These spikes, in turn, act as inputs for other neurons in the network. Neurons must execute this function across a diverse range of physiological conditions, often based on species-specific traits. Therefore, it is crucial to determine the extent to which findings can be extrapolated between species and, ultimately, to humans. In this study, we employed a multidisciplinary approach to pinpoint the factors accounting for the observed electrophysiological differences between mice and rats, the two species most used in experimental and computational research. After analyzing the morphological properties of their hippocampal CA1 pyramidal cells, we conducted a statistical comparison of rat and mouse electrophysiological features in response to somatic current injections. This analysis aimed to uncover the parameters underlying these distinctions. Using a well-established computational workflow, we created ten distinct single-cell computational models of mouse CA1 pyramidal neurons, ready to be used in a full-scale hippocampal circuit. By comparing their responses to a variety of somatic and synaptic inputs with those of rat models, we generated experimentally testable hypotheses regarding species-specific differences in ion channel distribution, kinetics, and the electrophysiological mechanisms underlying their distinct responses to synaptic inputs during the behaviorally relevant Gamma and Sharp-Wave rhythms.

Behavior of Calcium Phosphate-Chitosan-Collagen Composite Coating on AISI 304 for Orthopedic Applications.

Calcium phosphate/chitosan/collagen composite coating on AISI 304 stainless steel was investigated. Coatings were realized by galvanic coupling that occurs without an external power supply because it begins with the coupling between two metals with different standard electrochemical potentials. The process consists of the co-deposition of the three components with the calcium phosphate crystals incorporated into the polymeric composite of chitosan and collagen. Physical-chemical characterizations of the samples were executed to evaluate morphology and chemical composition. Morphological analyses have shown that the surface of the stainless steel is covered by the deposit, which has a very rough surface. XRD, Raman, and FTIR characterizations highlighted the presence of both calcium phosphate compounds and polymers. The coatings undergo a profound variation after aging in simulated body fluid, both in terms of composition and structure. The tests, carried out in simulated body fluid to scrutinize the corrosion resistance, have shown the protective behavior of the coating. In particular, the corrosion potential moved toward higher values with respect to uncoated steel, while the corrosion current density decreased. This good behavior was further confirmed by the very low quantification of the metal ions (practically absent) released in simulated body fluid during aging. Cytotoxicity tests using a pre-osteoblasts MC3T3-E1 cell line were also performed that attest the biocompatibility of the coating.

Effect of Polyhydroxyalkanoate (PHA) Concentration on Polymeric Scaffolds Based on Blends of Poly-L-Lactic Acid (PLLA) and PHA Prepared via Thermally Induced Phase Separation (TIPS).

Hybrid porous scaffolds composed of both natural and synthetic biopolymers have demonstrated significant improvements in the tissue engineering field. This study investigates for the first time the fabrication route and characterization of poly-L-lactic acid scaffolds blended with polyhydroxyalkanoate up to 30 wt%. The hybrid scaffolds were prepared by a thermally induced phase separation method starting from ternary solutions. The microstructure of the hybrid porous structures was analyzed by scanning electron microscopy and related to the blend composition. The porosity and the wettability of the scaffolds were evaluated through gravimetric and water contact angle measurements, respectively. The scaffolds were also characterized in terms of the surface chemical properties via Fourier transform infrared spectroscopy in attenuated total reflectance. The mechanical properties were analyzed through tensile tests, while the crystallinity of the PLLA/PHA scaffolds was investigated by differential scanning calorimetry and X-ray diffraction.

A dynamic air-liquid interface system for in vitro mimicking of the nasal mucosa.

The development of an in vitro 3D model for the growth of the nasal mucosa cells can improve the therapy and the study of pathological states for subjects with chronic airway conditions. We have previously characterized a system consisting of a scaffold with an internal channel and a perfusion bioreactor with two independent flows provided by an external and an internal circuit, respectively. In this paper, this system was designed as a model of the nasal cavity, in which cells, grown on the inner surface of the scaffold channel, would be in contact at the same time with both culture medium, supplied by the external circuit, and air, provided with the internal flow. To ensure adequate nutrient supply to the cells in the scaffold channel, the radial diffusion of the culture medium through the porous matrix was evaluated first in qualitative and, then, in quantitative terms, demonstrating the capability of the system to control the value and direction of this flux. As a preliminary study, the culture of epithelial cells in the scaffold channel is also discussed in static, maintaining the air-liquid interface condition for up to 3 weeks. Despite minor abnormalities, such as a gap between cell layers and some detachments from the scaffold, the scaffold ensured cell survival and growth during the experimental time.

Poly-l-Lactic Acid (PLLA)-Based Biomaterials for Regenerative Medicine: A Review on Processing and Applications.

Synthetic biopolymers are effective cues to replace damaged tissue in the tissue engineering (TE) field, both for in vitro and in vivo application. Among them, poly-l-lactic acid (PLLA) has been highlighted as a biomaterial with tunable mechanical properties and biodegradability that allows for the fabrication of porous scaffolds with different micro/nanostructures via various approaches. In this review, we discuss the structure of PLLA, its main properties, and the most recent advances in overcoming its hydrophobic, synthetic nature, which limits biological signaling and protein absorption. With this aim, PLLA-based scaffolds can be exposed to surface modification or combined with other biomaterials, such as natural or synthetic polymers and bioceramics. Further, various fabrication technologies, such as phase separation, electrospinning, and 3D printing, of PLLA-based scaffolds are scrutinized along with the in vitro and in vivo applications employed in various tissue repair strategies. Overall, this review focuses on the properties and applications of PLLA in the TE field, finally affording an insight into future directions and challenges to address an effective improvement of scaffold properties.

A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads.

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1ß exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-ß1 pathway. Notably, TGF-ß1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1ß exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment.

Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning calorimetry and X-ray diffraction analyses. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the Kef coating. The water resistance of Kefiran coating in distilled water at 37 °C evaluated on both PLA/Kef and P-PLA/Kef was carried out by gravimetric and morphological analyses. Finally, cell culture assays with embryonic fibroblast cells were conducted on selected hybrid scaffolds to compare the cell proliferation, morphology, and collagen production with PLA and P-PLA electrospun scaffolds. Based on the results, we can demonstrate that direct coating of PLA from Kef/water solutions is an effective approach to prepare hybrid scaffolds with tunable properties and that the plasma pre-treatment enhances the affinity between PLA and Kefiran. In vitro tests demonstrated the great potential of PLA/Kef hybrid scaffolds for skin tissue engineering.

Solution-Based Processing for Scaffold Fabrication in Tissue Engineering Applications: A Brief Review.

The fabrication of 3D scaffolds is under wide investigation in tissue engineering (TE) because of its incessant development of new advanced technologies and the improvement of traditional processes. Currently, scientific and clinical research focuses on scaffold characterization to restore the function of missing or damaged tissues. A key for suitable scaffold production is the guarantee of an interconnected porous structure that allows the cells to grow as in native tissue. The fabrication techniques should meet the appropriate requirements, including feasible reproducibility and time- and cost-effective assets. This is necessary for easy processability, which is associated with the large range of biomaterials supporting the use of fabrication technologies. This paper presents a review of scaffold fabrication methods starting from polymer solutions that provide highly porous structures under controlled process parameters. In this review, general information of solution-based technologies, including freeze-drying, thermally or diffusion induced phase separation (TIPS or DIPS), and electrospinning, are presented, along with an overview of their technological strategies and applications. Furthermore, the differences in the fabricated constructs in terms of pore size and distribution, porosity, morphology, and mechanical and biological properties, are clarified and critically reviewed. Then, the combination of these techniques for obtaining scaffolds is described, offering the advantages of mimicking the unique architecture of tissues and organs that are intrinsically difficult to design.

Novel dual-flow perfusion bioreactor for in vitro pre-screening of nanoparticles delivery: design, characterization and testing.

An advanced dual-flow perfusion bioreactor with a simple and compact design was developed and evaluated as a potential apparatus to reduce the gap between animal testing and drug administration to human subjects in clinical trials. All the experimental tests were carried out using an ad hoc Poly Lactic Acid (PLLA) scaffold synthesized via Thermally Induced Phase Separation (TIPS). The bioreactor shows a tunable radial flow throughout the microporous matrix of the scaffold. The radial perfusion was quantified both with permeability tests and with a mathematical model, applying a combination of Darcy's Theory, Bernoulli's Equation, and Poiseuille's Law. Finally, a diffusion test allowed to investigate the efficacy of the radial flow using Polymeric Fluorescent Nanoparticles (FNPs) mimicking drug/colloidal carriers. These tests confirmed the ability of our bioreactor to create a uniform distribution of particles inside porous matrices. All the findings candidate our system as a potential tool for drug pre-screening testing with a cost and time reduction over animal models.

Physical and biological properties of electrospun poly(d,l-lactide)/nanoclay and poly(d,l-lactide)/nanosilica nanofibrous scaffold for bone tissue engineering.

Electrospun scaffolds exhibiting high physical performances with the ability to support cell attachment and proliferation are attracting more and more scientific interest for tissue engineering applications. The inclusion of inorganic nanoparticles such as nanosilica and nanoclay into electrospun biopolymeric matrices can meet these challenging requirements. The silica and clay incorporation into polymeric nanofibers has been reported to enhance and improve the mechanical properties as well as the osteogenic properties of the scaffolds. In this work, for the first time, the physical and biological properties of polylactic acid (PLA) electrospun mats filled with different concentrations of nanosilica and nanoclay were evaluated and compared. The inclusion of the particles was evaluated through morphological investigations and Fourier transform infrared spectroscopy. The morphology of nanofibers was differently affected by the amount and kind of fillers and it was correlated to the viscosity of the polymeric suspensions. The wettability of the scaffolds, evaluated through wet contact angle measurements, slightly increased for both the nanocomposites. The crystallinity of the systems was investigated by differential scanning calorimetry highlighting the nucleating action of both nanosilica and nanoclay on PLA. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the fillers. Finally, cell culture assays with pre-osteoblastic cells were conducted on a selected composite scaffold in order to compare the cell proliferation and morphology with that of neat PLA scaffolds. Based on the results, we can convince that nanosilica and nanoclay can be both considered great potential fillers for electrospun systems engineered for bone tissue regeneration.