Unraveling the transcriptome profile of pulsed electromagnetic field stimulation in bone regeneration using a bioreactor-based investigation platform.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) sense and respond to biomechanical and biophysical stimuli, yet the involved signaling pathways are not fully identified. The clinical application of biophysical stimulation including pulsed electromagnetic field (PEMF) has gained momentum in musculoskeletal disorders and bone tissue engineering. METHODOLOGY: We herein aim to explore the role of PEMF stimulation in bone regeneration by developing trabecular bone-like tissues, and then, culturing them under bone-like mechanical stimulation in an automated perfusion bioreactor combined with a custom-made PEMF stimulator. After selecting the optimal cell seeding and culture conditions for inspecting the effects of PEMF on hMSCs, transcriptomic studies were performed on cells cultured under direct perfusion with and without PEMF stimulation. RESULTS: We were able to identify a set of signaling pathways and upstream regulators associated with PEMF stimulation and to distinguish those linked to bone regeneration. Our findings suggest that PEMF induces the immune potential of hMSCs by activating and inhibiting various immune-related pathways, such as macrophage classical activation and MSP-RON signaling in macrophages, respectively, while promoting angiogenesis and osteogenesis, which mimics the dynamic interplay of biological processes during bone healing. CONCLUSIONS: Overall, the adopted bioreactor-based investigation platform can be used to investigate the impact of PEMF stimulation on bone regeneration.

Adaptable test bench for ASTM-compliant permeability measurement of porous scaffolds for tissue engineering.

Intrinsic permeability describes the ability of a porous medium to be penetrated by a fluid. Considering porous scaffolds for tissue engineering (TE) applications, this macroscopic variable can strongly influence the transport of oxygen and nutrients, the cell seeding process, and the transmission of fluid forces to the cells, playing a crucial role in determining scaffold efficacy. Thus, accurately measuring the permeability of porous scaffolds could represent an essential step in their optimization process. In literature, several methods have been proposed to characterize scaffold permeability. Most of the currently adopted approaches to assess permeability limit their applicability to specific scaffold structures, hampering protocols standardization, and ultimately leading to incomparable results among different laboratories. The content of novelty of this study is in the proposal of an adaptable test bench and in defining a specific testing protocol, compliant with the ASTM International F2952-22 guidelines, for reliable and repeatable measurements of the intrinsic permeability of TE porous scaffolds. The developed permeability test bench (PTB) exploits the pump-based method, and it is composed of a modular permeability chamber integrated within a closed-loop hydraulic circuit, which includes a peristaltic pump and pressure sensors, recirculating demineralized water. A specific testing protocol was defined for characterizing the pressure drop associated with the scaffold under test, while minimizing the effects of uncertainty sources. To assess the operational capabilities and performance of the proposed test bench, permeability measurements were conducted on PLA scaffolds with regular (PS) and random (RS) micro-architecture and on commercial bovine bone matrix-derived scaffolds (CS) for bone TE. To validate the proposed approach, the scaffolds were as well characterized using an alternative test bench (ATB) based on acoustic measurements, implementing a blind randomized testing procedure. The consistency of the permeability values measured using both the test benches demonstrated the reliability of the proposed approach. A further validation of the PTB's measurement reliability was provided by the agreement between the measured permeability values of the PS scaffolds and the theory-based predicted permeability value. Once validated the proposed PTB, the performed measurements allowed the investigation of the scaffolds' transport properties. Samples with the same structure (guaranteed by the fused-deposition modeling technique) were characterized by similar permeability values, and CS and RS scaffolds showed permeability values in agreement with the values reported in the literature for bovine trabecular bone. In conclusion, the developed PTB and the proposed testing protocol allow the characterization of the intrinsic permeability of porous scaffolds of different types and dimensions under controlled flow regimes, representing a powerful tool in view of providing a reliable and repeatable framework for characterizing and optimizing scaffolds for TE applications.

Tailoring the Microarchitectures of 3D Printed Bone-like Scaffolds for Tissue Engineering Applications.

Material extrusion (MEX), commonly referred to as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a versatile and cost-effective technique to fabricate suitable scaffolds for tissue engineering. Driven by a computer-aided design input, specific patterns can be easily collected in an extremely reproducible and repeatable process. Referring to possible skeletal affections, 3D-printed scaffolds can support tissue regeneration of large bone defects with complex geometries, an open major clinical challenge. In this study, polylactic acid scaffolds were printed resembling trabecular bone microarchitecture in order to deal with morphologically biomimetic features to potentially enhance the biological outcome. Three models with different pore sizes (i.e., 500, 600, and 700 µm) were prepared and evaluated by means of micro-computed tomography. The biological assessment was carried out seeding SAOS-2 cells, a bone-like cell model, on the scaffolds, which showed excellent biocompatibility, bioactivity, and osteoinductivity. The model with larger pores, characterized by improved osteoconductive properties and protein adsorption rate, was further investigated as a potential platform for bone-tissue engineering, evaluating the paracrine activity of human mesenchymal stem cells. The reported findings demonstrate that the designed microarchitecture, better mimicking the natural bone extracellular matrix, favors a greater bioactivity and can be thus regarded as an interesting option for bone-tissue engineering.

Physiologic Response Evaluation of Human Foetal Osteoblast Cells within Engineered 3D-Printed Polylactic Acid Scaffolds.

Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field.

Scaffold-based bone tissue engineering in microgravity: potential, concerns and implications.

One of humanity's greatest challenges is space exploration, which requires an in-depth analysis of the data continuously collected as a necessary input to fill technological gaps and move forward in several research sectors. Focusing on space crew healthcare, a critical issue to be addressed is tissue regeneration in extreme conditions. In general, it represents one of the hottest and most compelling goals of the scientific community and the development of suitable therapeutic strategies for the space environment is an urgent need for the safe planning of future long-term manned space missions. Osteopenia is a commonly diagnosed disease in astronauts due to the physiological adaptation to altered gravity conditions. In order to find specific solutions to bone damage in a reduced gravity environment, bone tissue engineering is gaining a growing interest. With the aim to critically investigate this topic, the here presented review reports and discusses bone tissue engineering scenarios in microgravity, from scaffolding to bioreactors. The literature analysis allowed to underline several key points, such as the need for (i) biomimetic composite scaffolds to better mimic the natural microarchitecture of bone tissue, (ii) uniform simulated microgravity levels for standardized experimental protocols to expose biological materials to the same testing conditions, and (iii) improved access to real microgravity for scientific research projects, supported by the so-called democratization of space.

Biological Response to Bioinspired Microporous 3D-Printed Scaffolds for Bone Tissue Engineering.

The scaffold is a key element in the field of tissue engineering, especially when large defects or substitutions of pathological tissues or organs need to be clinically addressed. The expected outcome is strongly dependent on the cell-scaffold interaction and the integration with the surrounding biological tissue. Indeed, mimicking the natural extracellular matrix (ECM) of the tissue to be healed represents a further optimization that can limit a possible morphological mismatch between the scaffold and the tissue itself. For this aim, and referring to bone tissue engineering, polylactic acid (PLA) scaffolds were 3D printed with a microstructure inspired by the trabecular architecture and biologically evaluated by means of human osteosarcoma SAOS-2 cells. The cells were seeded on two types of scaffolds differing for the designed pore size (i.e., 400 and 600 µm), showing the same growth exponential trend found in the control and no significant alterations in the actin distribution. The microporous structure of the two tested samples enhanced the protein adsorption capability and mRNA expression of markers related to protein synthesis, proliferation, and osteoblast differentiation. Our findings demonstrate that 3D-printed scaffolds support the adhesion, growth, and differentiation of osteoblast-like cells and the microporous architecture, mimicking the natural bone hierarchical structure, and favoring greater bioactivity. These bioinspired scaffolds represent an interesting new tool for bone tissue engineering and regenerative medicine applications.

Analysis of the Chemical and Physical Environmental Aspects that Promoted the Spread of SARS-CoV-2 in the Lombard Area.

Recent works have demonstrated that particulate matter (PM) and specific meteorological conditions played an important role in the airborne transmission of the SARS-CoV-1 and MERS-CoV. These studies suggest that these parameters could influence the transmission of SARS-CoV-2. In the present investigation, we sought to investigate the association between air pollution, meteorological data, and the Lombardy region COVID-19 outbreak caused by SARS-CoV-2. We considered the number of detected infected people at the regional and provincial scale from February to March 2020. Air pollution data were collected over the Lombardy region, nominally, sulphur dioxide, ammonia, nitrogen dioxide, nitrogen monoxide, carbon monoxide, ozone, and suspended particulate matter measuring less than 10 µm (PM10) and less than 2.5 µm (PM2.5). Meteorological data have been collected over the same region for temperature, relative humidity, and wind speed. In this work, we evaluated the combined impact of environmental pollutants and climate conditions on the COVID-19 outbreak. The analysis evidenced a positive correlation between spatial distribution of COVID-19 infection cases with high concentrations of suspended particulate matter and a negative relationship with ozone. Moreover, suspended particulate matter concentration peaks in February correlated positively with infection peaks according to the virus incubation period. The obtained results suggested that seasonal weather conditions and concentration of air pollutants seemed to influence COVID-19 epidemics in Lombardy region.

3D Printing Decellularized Extracellular Matrix to Design Biomimetic Scaffolds for Skeletal Muscle Tissue Engineering.

Functional engineered muscles are still a critical clinical issue to be addressed, although different strategies have been considered so far for the treatment of severe muscular injuries. Indeed, the regenerative capacity of skeletal muscle (SM) results inadequate for large-scale defects, and currently, SM reconstruction remains a complex and unsolved task. For this aim, tissue engineered muscles should provide a proper biomimetic extracellular matrix (ECM) alternative, characterized by an aligned/microtopographical structure and a myogenic microenvironment, in order to promote muscle regeneration. As a consequence, both materials and fabrication techniques play a key role to plan an effective therapeutic approach. Tissue-specific decellularized ECM (dECM) seems to be one of the most promising material to support muscle regeneration and repair. 3D printing technologies, on the other side, enable the fabrication of scaffolds with a fine and detailed microarchitecture and patient-specific implants with high structural complexity. To identify innovative biomimetic solutions to develop engineered muscular constructs for the treatment of SM loss, the more recent (last 5 years) reports focused on SM dECM-based scaffolds and 3D printing technologies for SM regeneration are herein reviewed. Possible design inputs for 3D printed SM dECM-based scaffolds for muscular regeneration are also suggested.

Information-Driven Design as a Potential Approach for 3D Printing of Skeletal Muscle Biomimetic Scaffolds.

Severe muscle injuries are a real clinical issue that still needs to be successfully addressed. Tissue engineering can represent a potential approach for this aim, but effective healing solutions have not been developed yet. In this regard, novel experimental protocols tailored to a biomimetic approach can thus be defined by properly systematizing the findings acquired so far in the biomaterials and scaffold manufacturing fields. In order to plan a more comprehensive strategy, the extracellular matrix (ECM), with its properties stimulating neomyogenesis and vascularization, should be considered as a valuable biomaterial to be used to fabricate the tissue-specific three-dimensional structure of interest. The skeletal muscle decellularized ECM can be processed and printed, e.g., by means of stereolithography, to prepare bioactive and biomimetic 3D scaffolds, including both biochemical and topographical features specifically oriented to skeletal muscle regenerative applications. This paper aims to focus on the skeletal muscle tissue engineering sector, suggesting a possible approach to develop instructive scaffolds for a guided healing process.

3D printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and characterization.

Additive manufacturing for tissue engineering applications offers the possibility to design scaffolds characterized by a fine and detailed microarchitecture. Several fabrication technologies are currently available which allow to prepare tailored structures with a large selection of materials for restoring and healing tissues. However, 3D printed scaffolds are generally collected by assembling repetitive geometrical units or reproducing specific patterns in the layering direction, leading to a highly ordered architecture that does not mimic the morphology of the natural extracellular matrix (ECM), one of the main goals to be reached for an effective therapeutic approach. It is usually stated in the tissue engineering field that a scaffold has to be considered a temporary ECM, resembling all the peculiar features as close as possible and, in this regard, an ordered microstructure cannot be usually observed within biological tissues and organs. With the aim to overcame this limitation and offer a potential approach for bone tissue applications, the present study proposes a design methodology to fabricate 3D printed scaffolds characterized by a random microarchitecture which can be repeatedly reproduced thanks to the intrinsic controllable process of additive manufacturing. In this framework, four different models in polylactic acid were fabricated by means of fused deposition modelling, including a three-dimensional random distribution of spherical pores of 400, 500, and 600 µm for the first three cases, and a randomly varied distribution in the range 400-600 µm for the fourth case. A detailed assessment by means of microcomputed tomography and mechanical evaluation was then carried out in order to fully analyse the resulting scaffolds, providing both morphological and quantitative data.

Raman Spectroscopy and Aptamers for a Label-Free Approach: Diagnostic and Application Tools.

Raman spectroscopy is a powerful optical technique based on the inelastic scattering of incident light to assess the chemical composition of a sample, including biological ones. Medical diagnostic applications of Raman spectroscopy are constantly increasing to provide biochemical and structural information on several specimens, being not affected by water interference, and potentially avoiding the constraint of additional labelling procedures. New strategies have been recently developed to overcome some Raman limitations related, for instance, to the need to deal with an adequate quantity of the sample to perform a reliable analysis. In this regard, the use of metallic nanoparticles, the optimization of fiber optic probes, and other approaches can actually enhance the signal intensity compared to spontaneous Raman scattering. Moreover, to further increase the potential of this investigation technique, aptamers can be considered as a valuable means, being synthetic, short, single, or double-stranded oligonucleotides (RNAs or DNAs) that fold up into unique 3D structures to specifically bind to selected molecules, even at very low concentrations, and thus allowing an early diagnosis of a possible disease. Due to the paramount relevance of the topic, this review focuses on the main Raman spectroscopy techniques combined with aptamer arrays in the label-free mode, providing an overview on different applications to support healthcare management.

Thrombin Assessment on Nanostructured Label-Free Aptamer-Based Sensors: A Mapping Investigation via Surface-Enhanced Raman Spectroscopy.

Aptamers, synthetic single-stranded DNA or RNA molecules, can be regarded as a valuable improvement to develop novel ad hoc sensors to diagnose several clinical pathologies. Their intrinsic potential is related to the high specificity and sensitivity to the selected target biomarkers, being capable of detecting very low concentrations and thus allowing an early diagnosis of a possible disease. This kind of probe can be usefully integrated into a number of different devices in order to provide a reliable acquisition of the analyte and properly elaborate the related signal. The study presents the fabrication and characterization of a label-free aptamer sensor designed using a gold-coated silicon nanostructured substrate to map the target molecule by means of surface-enhanced Raman spectroscopy (SERS). As a proof, thrombin was used as a model at four different concentrations (i.e., 0.0873, 0.873, 8.73, and 87.3 nM). SERS mapping analysis was carried out considering each representative band of the aptamer-thrombin complex (centered at 822, 1140, and 1558 cm-1) and then combining them in order to acquire a comprehensive and unambiguous measure of the target. In both cases, a valuable correlation was evaluated, even if the first approach can suffer from some limitations in the third band related to lower definition of the characteristic peak compared to those in the other two bands.

Publisher Correction: Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats.

This corrects the article DOI: 10.1038/ncomms4562.

Natural polymeric microspheres for modulated drug delivery.

Microspheres can be regarded as a suitable platform for the development of ad hoc drug delivery systems, since the targeted release of a therapeutic agent can effectively contribute to support and improve a pharmacological protocol. However, several crucial factors related to the selection of materials, drugs and fabrication techniques should be critically analyzed in order to enhance the expected performance. Dealing with highly compatible materials, e.g. naturally-derived polymers and "green" reagents, can be a valid approach. For this aim, gelatin, chitosan and blend microspheres were produced by emulsion technique simply using distilled water and olive oil. Necessarily, due to the intrinsic instability of the selected materials in aqueous environment, microspheres were cross-linked with genipin, an extremely low cytotoxic agent, at three different concentration (i.e., 0.1, 0.5, 1% w/v). Collected microspheres were then loaded with methylene blue (MB), as drug model. Morphological analysis revealed homogeneous microspheres characterized by an average diameter comprised in the range 42-54µm. In vitro MB temporal delivery was assessed until complete release, which occurred in about 3days for gelatin and 30days for chitosan microspheres. Nanoindentation analysis was performed to evaluate how polymers and genipin influenced the mechanical properties of microspheres. Finally, the effect of released MB was investigated by means of chicken embryo chorioallantoic membrane assay, highlighting anti-angiogenic properties for gelatin differently from chitosan and blend microspheres.

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue-engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein-loading ability and biodegradability. Copyright © 2015 John Wiley & Sons, Ltd.