Early Impairment of Paracrine and Phenotypic Features in Resident Cardiac Mesenchymal Stromal Cells after Thoracic Radiotherapy.

Radiotherapy-induced cardiac toxicity and consequent diseases still represent potential severe late complications for many cancer survivors who undergo therapeutic thoracic irradiation. We aimed to assess the phenotypic and paracrine features of resident cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Resident CMSCs were isolated from a rat model of fractionated thoracic irradiation with accurate and clinically relevant heart dosimetry that developed delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 and 12 weeks after the end of radiotherapy and fully characterized at the transcriptional, paracrine, and functional levels. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. In particular, altered features included impaired cell migration and 3D growth and a and significant association of transcriptomic data with GO terms related to altered cytokine and growth factor signaling. Indeed, the altered paracrine profile of CMSCs derived from the group at the highest dose at the 12-week follow-up gave significantly reduced angiogenic support to endothelial cells and polarized macrophages toward a pro-inflammatory profile. Data collected in a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early paracrine and transcriptional alterations of the cardiac stroma may represent a dose- and time-dependent biological substrate for the delayed cardiac dysfunction phenotype observed in vivo.

Multiscale Mechanical Characterization of Polyether-2-ketone (PEKK) for Biomedical Application.

Polyether-ether-2-ketone (PEKK) is a high-performance thermoplastic polymer used in various fields, from aerospace to medical applications, due to its exceptional mechanical and thermal properties. Nonetheless, the mechanical behavior of 3D-printed PEKK still deserves to be more thoroughly investigated, especially in view of its production by 3D printing, where mechanical properties measured at different scales are likely to be correlated to one another and to all play a major role in determining biomechanical properties, which include mechanical strength on one side and osteointegration ability on the other side. This work explores the mechanical behavior of 3D-printed PEKK through a multiscale approach, having performed both nanoindentation tests and standard tensile and compression tests, where a detailed view of strain distribution was achieved through Digital Image Correlation (DIC) techniques. Furthermore, for specimens tested up to failure, their fractured surfaces were analyzed through Scanning Electron Microscopy (SEM) to clearly outline fracture modes. Additionally, the internal structure of 3D-printed PEKK was explored through Computed Tomography (CT) imaging, providing a three-dimensional view of the internal structure and the presence of voids and other imperfections. Finally, surface morphology was analyzed through confocal microscopy. The multiscale approach adopted in the present work offers information about the global and local behavior of the PEKK, also assessing its material properties down to the nanoscale. Due to its novelty as a polymeric material, no previous studies have approached a multiscale analysis of 3D-printed PEKK. The findings of this study contribute to a comprehensive understanding of 3D-printed PEKK along with criteria for process optimization in order to customize its properties to meet specific application requirements. This research not only advances the knowledge of PEKK as a 3D-printing material but also provides insights into the multifaceted nature of multiscale material characterization.

Short-Term Effects of 3D-Printed Occlusal Splints and Conventional Splints on Sleep Bruxism Activity: EMG-ECG Night Recordings of a Sample of Young Adults.

(1) Background: This study aims to compare the effects of 3D-printed splints and conventional manufactured splints on sleep bruxism (SB) EMG activity. (2) Methods: Twenty-six patients (19 M, 7 F, 25.8 ± 2.6 years) were randomly allocated to a study group (3D splints) and a control group (conventional manufactured splints) and followed for a period of three months with night EMG-ECG recordings. Samples of the involved materials were analyzed for nanoindentation. The outcomes of interest considered were the overall SB index, the total amount of surface masseter muscle activity (sMMA), and general and SB-related phasic and tonic contractions. A statistical evaluation was performed with a confidence interval (CI) between 2.5% and 97.5%. (3) Results: Differences between groups with OAs were observed for general tonic contraction (p = 0.0009), while differences between recording times were observed for general phasic contractions (p = 0.002) and general tonic contractions (p = 0.00001). Differences between recording times were observed for the total amount of sMMA (p = 0.01), for general phasic contractions (p = 0.0001), and for general tonic contractions (p = 0.000009) during night recordings without OAs. (4) Conclusions: Three-dimensional splints seem to have a higher impact on SB-related electromyographic activity but not on the overall sleep bruxism index. The more regular surfaces offered by 3D splints could be related to phasic contraction stabilization.

Development and validation of cryopreserved or freeze-dried decellularized human dermis for transplantation.

For decades, dermal tissue grafts have been used in various regenerative, reconstructive, and augmentative procedures across the body. To eliminate antigenicity and immunogenic response while still preserving the individual components and collective structural integrity of the extracellular matrix (ECM), dermis can be decellularized. Acellular dermal matrix (ADM) products like such are produced to accurately serve diverse clinical purposes. The aim of the present study is to evaluate the efficacy of a novel decellularization protocol of the human dermis, which eliminates residual human genetic material without compromising the biomechanical integrity and collagenous content of the tissue. Moreover, a freeze-drying protocol was validated. The results showed that though our decellularization protocol, human dermis can be decellularized obtaining a biocompatible matrix. The procedure is completely realized in GMP aseptic condition, avoiding tissue terminal sterilization.

Decellularized cryopreserved human pericardium: a validation study towards tissue bank practice.

Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium.

Could light-curing time, post-space region and cyclic fatigue affect the nanomechanical behavior of a dual-curing cement for fiber post luting?

OBJECTIVE: To evaluate the effects of curing time, post-space region and cyclic fatigue on the micromechanical properties of a fiber-post luting cement. The null hypotheses were that (1) curing time, (2) fatigue and (3) post-space region does not affect the nanoindentation modulus and hardness of the dual-curing cement. MATERIALS AND METHODS: 48 premolars were endodontically treated and a class I cavity and 8 mm deep post space was prepared. Fiber posts were luted with a universal, dualized adhesive system and a dual-curing cement following manufacturer's instructions. Specimens were divided into three groups (16 specimens for each group) according to light-curing time (no light-curing, 20 s light-curing and 120 s light-curing), which was performed with a LED lamp at 1000 mW/cm 2. The coronal part of the cavity was restored using a nano-filled resin composite. After 24 h, 8 specimens for each group were randomly extract in order to undergo to fatigue test in wet condition through a chewing simulator, while the other specimens were kept in distilled water as benchmark. All the restored teeth were then sectioned in 1 mm thick slices perpendicularly to the fiber post axis. Specimen slices were classified in coronal and apical to be tested through a nanoindenter. Data were analyzed through Kruskal-Wallis test with a significance level of 1%, in order to evaluate the influence of treatments (i.e., curing time and cyclic loading) on the micromechanical properties of the tested luting cement. RESULTS: Both fatigue and curing time significantly influenced nanoindentation modulus and hardness of dual-curing cement (p < 0.01). No significant differences were reported for post space region. A significant interaction was found among the analyzed factors (p < 0.01). SIGNIFICANCE: 120 s light-curing time is recommended in order to achieve optimal mechanical proprieties, independently from post space region and cyclic fatigue. As matter of fact, 120 s light-curing allowed to prevent strain hardening induced by the fatigue simulation.

Physical Characterization of Bismuth Oxide Nanoparticle Based Ceramic Composite for Future Biomedical Application.

Employment and the effect of eco-friendly bismuth oxide nanoparticles (BiONPs) in bio-cement were studied. The standard method was adopted to prepare BiONPs-composite. Water was adopted for dispersing BiONPs in the composite. A representative batch (2 wt. % of BiONPs) was prepared without water to study the impact of water on composite properties. For each batch, 10 samples were prepared and tested. TGA (thermogravimetric analysis) performed on composite showed 0.8 wt. % losses in samples prepared without water whereas, maximum 2 wt. % weight losses observed in the water-based composite. Presence of BiONPs resulted in a decrease in depth of curing. Three-point bending flexural strength decreased for increasing BiONPs content. Comparative study between 2 wt. % samples with and without water showed 10.40 (±0.91) MPa and 28.45 (±2.50) MPa flexural strength values, respectively, indicating a significant (p < 0.05) increase of the mechanical properties at the macroscale. Nanoindentation revealed that 2 wt. % without water composites showed significant (p < 0.05) highest nanoindentation modulus 26.4 (±1.28) GPa and hardness 0.46 (±0.013) GPa. Usage of water as dispersion media was found to be deleterious for the overall characteristics of the composite but, at the same time, the BiONPs acted as a very promising filler that can be used in this class of composites.

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs.

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro. In tissue engineering applications, bioreactors have become fundamental and effective tools for providing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on decellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation.

Decellularized Human Dermal Matrix as a Biological Scaffold for Cardiac Repair and Regeneration.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-µm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE.

Bread-Derived Bioactive Porous Scaffolds: An Innovative and Sustainable Approach to Bone Tissue Engineering.

In recent years, bioactive glasses gained increasing scientific interest in bone tissue engineering due to their capability to chemically bond with the host tissue and to induce osteogenesis. As a result, several efforts have been addressed to use bioactive glasses in the production of three-dimensional (3D) porous scaffolds for bone regeneration. In this work, we creatively combine typical concepts of porous glass processing with those of waste management and propose, for the first time, the use of bread as a new sacrificial template for the fabrication of bioactive scaffolds. Preliminary SEM investigations performed on stale bread from industrial wastes revealed a suitable morphology characterized by an open-cell 3D architecture, which is potentially able to allow tissue ingrowth and vascularization. Morphological features, mechanical performances and in vitro bioactivity tests were performed in order to evaluate the properties of these new "sustainable" scaffolds for bone replacement and regeneration. Scaffolds with total porosity ranging from 70 to 85 vol% and mechanical strength comparable to cancellous bone were obtained. Globular hydroxyapatite was observed to form on the surface of the scaffolds after just 48-h immersion in simulated body fluid. The results show great promise and suggest the possibility to use bread as an innovative and inexpensive template for the development of highly-sustainable bone tissue engineering approaches.

Application of 3D Printing Technology for Design and Manufacturing of Customized Components for a Mechanical Stretching Bioreactor.

Three-dimensional (3D) printing represents a key technology for rapid prototyping, allowing easy, rapid, and low-cost fabrication. In this work, 3D printing was applied for the in-house production of customized components of a mechanical stretching bioreactor with potential application for cardiac tissue engineering and mechanobiology studies. The culture chamber housing and the motor housing were developed as functional permanent parts, aimed at fixing the culture chamber position and at guaranteeing motor watertightness, respectively. Innovative sample holder prototypes were specifically designed and 3D-printed for holding thin and soft biological samples during cyclic stretch culture. The manufactured components were tested in-house and in a cell biology laboratory. Moreover, tensile tests and finite element analysis were performed to investigate the gripping performance of the sample holder prototypes. All the components showed suitable performances in terms of design, ease of use, and functionality. Based on 3D printing, the bioreactor optimization was completely performed in-house, from design to fabrication, enabling customization freedom, strict design-to-prototype timing, and cost and time effective testing, finally boosting the bioreactor development process.

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.