Publications by authors named "Marco Costantini"

50 Publications

Nanotechnology-Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID-19 Vaccines.

Small Methods 2021 Jul 28:2100402. Epub 2021 Jul 28.

Department of Biosystems and Soft Matter Institute of Fundamental Technological Research Polish Academy of Sciences ul. Pawińskiego 5B Warsaw 02-106 Poland.

In recent years, the main quest of science has been the pioneering of the groundbreaking biomedical strategies needed for achieving a personalized medicine. Ribonucleic acids (RNAs) are outstanding bioactive macromolecules identified as pivotal actors in regulating a wide range of biochemical pathways. The ability to intimately control the cell fate and tissue activities makes RNA-based drugs the most fascinating family of bioactive agents. However, achieving a widespread application of RNA therapeutics in humans is still a challenging feat, due to both the instability of naked RNA and the presence of biological barriers aimed at hindering the entrance of RNA into cells. Recently, material scientists' enormous efforts have led to the development of various classes of nanostructured carriers customized to overcome these limitations. This work systematically reviews the current advances in developing the next generation of drugs based on nanotechnology-assisted RNA delivery. The features of the most used RNA molecules are presented, together with the development strategies and properties of nanostructured vehicles. Also provided is an in-depth overview of various therapeutic applications of the presented systems, including coronavirus disease vaccines and the newest trends in the field. Lastly, emerging challenges and future perspectives for nanotechnology-mediated RNA therapies are discussed.
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http://dx.doi.org/10.1002/smtd.202100402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420172PMC
July 2021

Skin-Friction-Based Identification of the Critical Lines in a Transonic, High Reynolds Number Flow via Temperature-Sensitive Paint.

Sensors (Basel) 2021 Jul 28;21(15). Epub 2021 Jul 28.

National Research Council (CNR), Institute of Marine Engineering, Via di Vallerano 139, I-00128 Rome, Italy.

In this contribution, three methodologies based on temperature-sensitive paint (TSP) data were further developed and applied for the optical determination of the critical locations of flow separation and reattachment in compressible, high Reynolds number flows. The methodologies rely on skin-friction extraction approaches developed for low-speed flows, which were adapted in this work to study flow separation and reattachment in the presence of shock-wave/boundary-layer interaction. In a first approach, skin-friction topological maps were obtained from time-averaged surface temperature distributions, thus enabling the identification of the critical lines as converging and diverging skin-friction lines. In the other two approaches, the critical lines were identified from the maps of the propagation celerity of temperature perturbations, which were determined from time-resolved TSP data. The experiments were conducted at a freestream Mach number of 0.72 and a chord Reynolds number of 9.7 million in the Transonic Wind Tunnel Göttingen on a VA-2 supercritical airfoil model, which was equipped with two exchangeable TSP modules specifically designed for transonic, high Reynolds number tests. The separation and reattachment lines identified via the three different TSP-based approaches were shown to be in mutual agreement, and were also found to be in agreement with reference experimental and numerical data.
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http://dx.doi.org/10.3390/s21155106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348098PMC
July 2021

Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes.

Int J Mol Sci 2021 May 30;22(11). Epub 2021 May 30.

Fondazione Istituto Nazionale di Genetica Molecolare, 20122 Milan, Italy.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4 and CD8 lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8 cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4 and CD8 cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional system, able to recapitulate the rheological and stiffness properties of the tumor ECM.
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http://dx.doi.org/10.3390/ijms22115862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198248PMC
May 2021

Recent advances in bioprinting technologies for engineering cardiac tissue.

Mater Sci Eng C Mater Biol Appl 2021 May 25;124:112057. Epub 2021 Mar 25.

Research Center "E. Piaggio" and Department of Information Engineering, University of Pisa, Largo Lucio Lazzarino 1, 56122 Pisa, Italy. Electronic address:

Annually increasing incidence of cardiac-related disorders and cardiac tissue's minimal regenerative capacity have motivated the researchers to explore effective therapeutic strategies. In the recent years, bioprinting technologies have witnessed a great wave of enthusiasm and have undergone steady advancements over a short period, opening the possibilities for recreating engineered functional cardiac tissue models for regenerative and diagnostic applications. With this perspective, the current review delineates recent developments in the sphere of engineered cardiac tissue fabrication, using traditional and advanced bioprinting strategies. The review also highlights different printing ink formulations, available cellular opportunities, and aspects of personalized medicines in the context of cardiac tissue engineering and bioprinting. On a concluding note, current challenges and prospects for further advancements are also discussed.
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http://dx.doi.org/10.1016/j.msec.2021.112057DOI Listing
May 2021

Cochlear Synaptopathy due to Mutations in otof Gene May Result in Stable Mild Hearing Loss and Severe Impairment of Speech Perception.

Ear Hear 2021 Apr 27. Epub 2021 Apr 27.

Department of Neurosciences, University of Padua, Padua, Italy Audiology Service, Santi Giovanni e Paolo Hospital, Venezia, Italy Servicio de Genética, Hospital Universitario Ramón y Cajal, Madrid, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.

Objectives: Congenital profound hearing loss with preserved cochlear outer hair cell activity (otoacoustic emissions and cochlear microphonic) is the most common phenotype associated with mutations in the OTOF gene. The aim of this study was to investigate the pathophysiological mechanisms behind the auditory dysfunction in five patients (2 adults and 3 children) carrying biallelic mutations in OTOF, who showed an uncommon phenotype of mild hearing impairment associated with severe difficulties in speech perception and delay of language development.

Design: Patients underwent audiometric assessment with pure-tone and speech perception evaluation, and otoacoustic emissions and auditory brainstem response recording. Cochlear potentials were recorded in all subjects through transtympanic electrocochleography in response to clicks delivered in the free field from 120 to 60 dB peak equivalent SPL and were compared to recordings obtained from 20 normally hearing controls and from eight children with profound deafness due to mutations in the OTOF gene. Three patients out of five underwent unilateral cochlear implantation. Speech perception measures and electrically evoked auditory nerve potentials were obtained within 1 year of cochlear implant use.

Results: Pathogenic mutations in the two alleles of OTOF were found in all five patients, and five novel mutations were identified. Hearing thresholds indicated mild hearing loss in four patients and moderate hearing loss in one. Distortion product otoacoustic emissions were recorded in all subjects, whereas auditory brainstem responses were absent in all but two patients, who showed a delayed wave V in one ear. In electrocochleography recordings, cochlear microphonics and summating potentials showed normal latency and peak amplitude, consistently with preservation of both outer and inner hair cell activity. In contrast, the neural compound action potential recorded in normally hearing controls was replaced by a prolonged, low-amplitude negative response. No differences in cochlear potentials were found between OTOF subjects showing mild or profound hearing loss. Electrical stimulation through the cochlear implant improved speech perception and restored synchronized auditory nerve responses in all cochlear implant recipients.

Conclusions: These findings indicate that disordered synchrony in auditory fiber activity underlies the impairment of speech perception in patients carrying biallelic mutations in OTOF gene who show a stable phenotype of mild hearing loss. Abnormal nerve synchrony with preservation of hearing sensitivity is consistent with selective impairment of vesicle replenishment at the ribbon synapses with relative preservation of synaptic exocytosis. Cochlear implants are effective in restoring speech perception and synchronous activation of the auditory pathway by directly stimulating auditory fibers.
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http://dx.doi.org/10.1097/AUD.0000000000001052DOI Listing
April 2021

Recent advances in bioprinting technologies for engineering different cartilage-based tissues.

Mater Sci Eng C Mater Biol Appl 2021 Apr 4;123:112005. Epub 2021 Mar 4.

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India. Electronic address:

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.
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http://dx.doi.org/10.1016/j.msec.2021.112005DOI Listing
April 2021

Oxygen releasing materials: Towards addressing the hypoxia-related issues in tissue engineering.

Mater Sci Eng C Mater Biol Appl 2021 Mar 23;122:111896. Epub 2021 Jan 23.

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India. Electronic address:

Manufacturing macroscale cell-laden architectures is one of the biggest challenges faced nowadays in the domain of tissue engineering. Such living constructs, in fact, pose strict requirements for nutrients and oxygen supply that can hardly be addressed through simple diffusion in vitro or without a functional vasculature in vivo. In this context, in the last two decades, a substantial amount of work has been carried out to develop smart materials that could actively provide oxygen-release to contrast local hypoxia in large-size constructs. This review provides an overview of the currently available oxygen-releasing materials and their synthesis and mechanism of action, highlighting their capacities under in vitro tissue cultures and in vivo contexts. Additionally, we also showcase an emerging concept, herein termed as "living materials as releasing systems", which relies on the combination of biomaterials with photosynthetic microorganisms, namely algae, in an "unconventional" attempt to supply the damaged or re-growing tissue with the necessary supply of oxygen. We envision that future advances focusing on tissue microenvironment regulated oxygen-supplying materials would unlock an untapped potential for generating a repertoire of anatomic scale, living constructs with improved cell survival, guided differentiation, and tissue-specific biofunctionality.
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http://dx.doi.org/10.1016/j.msec.2021.111896DOI Listing
March 2021

Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration.

EMBO Mol Med 2021 Mar 15;13(3):e12778. Epub 2021 Feb 15.

Department of Biology, Rome University Tor Vergata, Rome, Italy.

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions-pivotal aspects for cell survival and muscle contractile functionalities-together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.
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http://dx.doi.org/10.15252/emmm.202012778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933978PMC
March 2021

organized neovascularization induced by 3D bioprinted endothelial-derived extracellular vesicles.

Biofabrication 2021 04 7;13(3). Epub 2021 Apr 7.

Istituto Nazionale Genetica Molecolare INGM 'Romeo ed Enrica Invernizzi', Milan, Italy.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. Aftersubcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.
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http://dx.doi.org/10.1088/1758-5090/abdacfDOI Listing
April 2021

Bactericidal antibodies against hypervirulent C field strains following MenC-CRM or MenACWY-CRM priming and MenACWY-CRM booster in children.

Hum Vaccin Immunother 2021 05 16;17(5):1442-1449. Epub 2020 Dec 16.

Clinical Research and Development, GSK, Siena, Italy.

An increase in invasive meningococcal disease (IMD) incidence was observed in Tuscany in 2015/2016, mainly due to hypervirulent clonal complex (cc) 11 strains. In a analysis, we assessed bactericidal activity of antibodies in sera from children primed with MenACWY-CRM or MenC-CRM conjugate vaccines and receiving a MenACWY-CRM booster dose against 5 meningococcal C (MenC) strains isolated from IMD cases. Sera collected from 90 infants/toddlers who participated in a phase III, open-label study (NCT00667602) and its extension (NCT01345721) were tested by serum bactericidal activity assay with human complement (hSBA). Children were primed with either MenACWY-CRM at 6-8 and 12 months of age (group 2_MenACWY; N = 30), MenACWY-CRM (group 1_MenACWY; N = 30), or MenC-CRM at 12 months of age (group 1_MenC; N = 30); all received MenACWY-CRM booster dose at 22-45 months of age. Four tested strains (FI001-FI004) were C:P1.5-1,10-8:F3-6:ST-11 (cc11) and 1 (FI005) was C:P1.7-4,14-6:F3-9:ST-1031 (cc334). Overall, immune responses tended to be higher against Fl002-FI004 than Fl001 and Fl005. Geometric mean titers were high in group 2_MenACWY (range: 94.8 [FI005]-588.1 [FI004]) and very high post-boosting with MenACWY-CRM in all groups (176.9 [FI005]-3911.0 [FI004]). Seroresponse rates tended to be higher in group 1_MenC (33.3% [FI005]-93.3% [FI004]) than in group 1_MenACWY (16.7% [FI005]-73.3% [FI004]). Irrespective of strains tested or the identity/number of priming doses, ≥96.7% of children had hSBA titers ≥1:8 post-MenACWY-CRM booster dose. MenACWY-CRM and MenC-CRM elicited bactericidal antibodies and immunological memory against hypervirulent cc11 and cc334 MenC strains responsible for IMD outbreaks.
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http://dx.doi.org/10.1080/21645515.2020.1833578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078732PMC
May 2021

Extrusion and Microfluidic-based Bioprinting to Fabricate Biomimetic Tissues and Organs.

Adv Mater Technol 2020 Aug 26;5(8). Epub 2020 May 26.

Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, CA 90095, USA.

Next generation engineered tissue constructs with complex and ordered architectures aim to better mimic the native tissue structures, largely due to advances in three-dimensional (3D) bioprinting techniques. Extrusion bioprinting has drawn tremendous attention due to its widespread availability, cost-effectiveness, simplicity, and its facile and rapid processing. However, poor printing resolution and low speed have limited its fidelity and clinical implementation. To circumvent the downsides associated with extrusion printing, microfluidic technologies are increasingly being implemented in 3D bioprinting for engineering living constructs. These technologies enable biofabrication of heterogeneous biomimetic structures made of different types of cells, biomaterials, and biomolecules. Microfluiding bioprinting technology enables highly controlled fabrication of 3D constructs in high resolutions and it has been shown to be useful for building tubular structures and vascularized constructs, which may promote the survival and integration of implanted engineered tissues. Although this field is currently in its early development and the number of bioprinted implants is limited, it is envisioned that it will have a major impact on the production of customized clinical-grade tissue constructs. Further studies are, however, needed to fully demonstrate the effectiveness of the technology in the lab and its translation to the clinic.
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http://dx.doi.org/10.1002/admt.201901044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567134PMC
August 2020

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities.

Front Physiol 2020 16;11:553198. Epub 2020 Sep 16.

Department of Biology, University of Rome Tor Vergata, Rome, Italy.

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy.
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http://dx.doi.org/10.3389/fphys.2020.553198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526461PMC
September 2020

Photocurable Biopolymers for Coaxial Bioprinting.

Methods Mol Biol 2021 ;2147:45-54

Università Campus Bio-Medico di Roma, Rome, Italy.

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication-in one step-of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition.To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers-namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen-that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications.
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http://dx.doi.org/10.1007/978-1-0716-0611-7_4DOI Listing
March 2021

Predicted long-term antibody persistence for a tick-borne encephalitis vaccine: results from a modeling study beyond 10 years after a booster dose following different primary vaccination schedules.

Hum Vaccin Immunother 2020 09 17;16(9):2274-2279. Epub 2020 Jan 17.

GSK , Siena, Italy.

In tick-borne encephalitis (TBE)-endemic regions, long-term vaccination programs are efficient in preventing the disease. A booster dose of a polygeline-free inactivated TBE vaccine (, GSK), administered approximately 3 years post-primary vaccination according to 1 of 3 licensed vaccination schedules in adults and adolescents, resulted in antibody persistence for 10 years post-boosting. We used different power-law models (PLMs) to predict long-term persistence of anti-TBE virus neutralization test (NT) antibody titers over a period of 20 years post-booster dose, based on individual antibody NT titers measured for 10 years post-booster vaccination. The PLMs were fitted on pooled data for all vaccine schedules. A mean NT titer of 261 (95% prediction interval: 22-3096), considerably above the accepted threshold of protection (NT titers ≥10), was predicted 20 years post-booster vaccination with TBE vaccine. Our modeled data suggest that the intervals of booster doses could be increased without compromising protection against TBE.
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http://dx.doi.org/10.1080/21645515.2019.1700712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553683PMC
September 2020

One-year antibody persistence and safety of a 4-dose schedule of MenACWY-CRM in healthy infants from South Korea.

Clin Exp Vaccine Res 2019 Jul 31;8(2):94-102. Epub 2019 Jul 31.

GSK, Siena, Italy.

Purpose: Results from a post-marketing study to generate evidence on 1-year antibody persistence and safety following vaccination of infants from South Korea with the quadrivalent meningococcal conjugate vaccine MenACWY-CRM.

Materials And Methods: In this phase IV, open-label, multi-center study (NCT02446691), 128 infants received MenACWY-CRM at ages 2, 4, 6, and 12 months. One-year antibody persistence following the full vaccination course was evaluated (primary objective) for the four meningococcal serogroups (Men) by serum bactericidal activity assay using human or rabbit complement (hSBA/rSBA). Immune responses at 1-month post-vaccination and safety were also assessed.

Results: The percentage of children with hSBA titers ≥8 ranged between 94% (MenA) and 100% (MenY/W) 1-month post-vaccination, and from 39% (MenA) to 89% (MenY) 1-year post-vaccination. At least 99% and 92% of children had rSBA titers ≥8 and ≥128 against each meningococcal serogroup, 1-month post-vaccination. One-year post-vaccination, the percentage of children with rSBA titers ≥8 and ≥128 ranged from 54% (MenC) to 99% (MenA) and from 30% (MenC) to 98% (MenA). Geometric mean titers declined from 1-month to 1-year post-vaccination, when they varied between 6.8 (MenA) and 53.6 (MenW) by hSBA and between 17.2 (MenC) and 2,269.5 (MenA) by rSBA. At least one solicited and unsolicited adverse event was reported for 79% and 66% of children. Of 36 serious adverse events reported, none were vaccination-related.

Conclusion: Antibody persistence (hSBA/rSBA titers ≥8) was determined in 39%-99% of children 1 year after a 4-dose MenACWY-CRM series during infancy, with an acceptable clinical safety profile.
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http://dx.doi.org/10.7774/cevr.2019.8.2.94DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689500PMC
July 2019

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head.

Biofabrication 2019 07 1;11(4):044101. Epub 2019 Jul 1.

Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.
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http://dx.doi.org/10.1088/1758-5090/ab2622DOI Listing
July 2019

3D Bioprinting in Skeletal Muscle Tissue Engineering.

Small 2019 06 23;15(24):e1805530. Epub 2019 Apr 23.

Center for Minimally Invasive Therapeutics (C-MIT), Department of Radiological Sciences, University of California, Los Angeles, CA, 90095, USA.

Skeletal muscle tissue engineering (SMTE) aims at repairing defective skeletal muscles. Until now, numerous developments are made in SMTE; however, it is still challenging to recapitulate the complexity of muscles with current methods of fabrication. Here, after a brief description of the anatomy of skeletal muscle and a short state-of-the-art on developments made in SMTE with "conventional methods," the use of 3D bioprinting as a new tool for SMTE is in focus. The current bioprinting methods are discussed, and an overview of the bioink formulations and properties used in 3D bioprinting is provided. Finally, different advances made in SMTE by 3D bioprinting are highlighted, and future needs and a short perspective are provided.
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http://dx.doi.org/10.1002/smll.201805530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570559PMC
June 2019

3D Printing of Thermoresponsive Polyisocyanide (PIC) Hydrogels as Bioink and Fugitive Material for Tissue Engineering.

Polymers (Basel) 2018 May 21;10(5). Epub 2018 May 21.

Faculty of Materials Science and Engineering, Warsaw University of Technology, 141 Woloska str., 02-507 Warsaw, Poland.

Despite the rapid and great developments in the field of 3D hydrogel printing, a major ongoing challenge is represented by the development of new processable materials that can be effectively used for bioink formulation. In this work, we present an approach to 3D deposit, a new class of fully-synthetic, biocompatible PolyIsoCyanide (PIC) hydrogels that exhibit a reverse gelation temperature close to physiological conditions (37 °C). Being fully-synthetic, PIC hydrogels are particularly attractive for tissue engineering, as their properties-such as hydrogel stiffness, polymer solubility, and gelation kinetics-can be precisely tailored according to process requirements. Here, for the first time, we demonstrate the feasibility of both 3D printing PIC hydrogels and of creating dual PIC-Gelatin MethAcrylate (GelMA) hydrogel systems. Furthermore, we propose the use of PIC as fugitive hydrogel to template structures within GelMA hydrogels. The presented approach represents a robust and valid alternative to other commercial thermosensitive systems-such as those based on Pluronic F127-for the fabrication of 3D hydrogels through additive manufacturing technologies to be used as advanced platforms in tissue engineering.
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http://dx.doi.org/10.3390/polym10050555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415434PMC
May 2018

3D bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering.

Biofabrication 2019 05 3;11(3):035016. Epub 2019 May 3.

Warsaw University of Technology, Faculty of Materials Science and Engineering, 02-507 Warsaw, Poland.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing β-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.
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http://dx.doi.org/10.1088/1758-5090/ab15cbDOI Listing
May 2019

3D-Printing of Functionally Graded Porous Materials Using On-Demand Reconfigurable Microfluidics.

Angew Chem Int Ed Engl 2019 Jun 29;58(23):7620-7625. Epub 2019 Apr 29.

Institute of Physical Chemistry-, Polish Academy of Sciences, Ul. Kasprzaka 44/52, 01224, Warsaw, Poland.

Tailoring the morphology of macroporous structures remains one of the biggest challenges in material synthesis. Herein, we present an innovative approach for the fabrication of custom macroporous materials in which pore size varies throughout the structure by up to an order of magnitude. We employed a valve-based flow-focusing junction (vFF) in which the size of the orifice can be adjusted in real-time (within tens of milliseconds) to generate foams with on-line controlled bubble size. We used the junction to fabricate layered and smoothly graded porous structures with pore size varying in the range of 80-800 μm. Additionally, we mounted the vFF on top of an extrusion printer and 3D-printed constructs characterized by a predefined 3D geometry and a controlled, spatially varying internal porous architecture, such as a model of a bone. The presented technology opens new possibilities in macroporous material synthesis with potential applications ranging from tissue engineering to aerospace industry and construction.
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http://dx.doi.org/10.1002/anie.201900530DOI Listing
June 2019

Tendon Tissue Engineering: Effects of Mechanical and Biochemical Stimulation on Stem Cell Alignment on Cell-Laden Hydrogel Yarns.

Adv Healthc Mater 2019 04 6;8(7):e1801218. Epub 2019 Feb 6.

Faculty of Material Science and Engineering, Warsaw University of Technology, Warsaw, 02-507, Poland.

Fiber-based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro-environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell-laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM-MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP-12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell-laden aligned system can be used for engineering of scaffolds for tendon regeneration.
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http://dx.doi.org/10.1002/adhm.201801218DOI Listing
April 2019

Co-axial wet-spinning in 3D bioprinting: state of the art and future perspective of microfluidic integration.

Biofabrication 2018 11 9;11(1):012001. Epub 2018 Nov 9.

Department of Chemistry, University of Rome 'La Sapienza', 00185 Rome, Italy. Warsaw University of Technology, Faculty of Materials Science and Engineering, Warsaw, Poland. Institute of Physical Chemistry, Polish Academy of Sciences, 01224 Warsaw, Poland.

Nowadays, 3D bioprinting technologies are rapidly emerging in the field of tissue engineering and regenerative medicine as effective tools enabling the fabrication of advanced tissue constructs that can recapitulate in vitro organ/tissue functions. Selecting the best strategy for bioink deposition is often challenging and time consuming process, as bioink properties-in the first instance, rheological and gelation-strongly influence the suitable paradigms for its deposition. In this short review, we critically discuss one of the available approaches used for bioprinting-namely co-axial wet-spinning extrusion. Such a deposition system, in fact, demonstrated to be promising in terms of printing resolution, shape fidelity and versatility when compared to other methods. An overview of the performances of co-axial technology in the deposition of cellularized hydrogel fibres is discussed, highlighting its main features. Furthermore, we show how this approach allows (i) to decouple the printing accuracy from bioink rheological behaviour-thus notably simplifying the development of new bioinks-and (ii) to build heterogeneous multi-materials and/or multicellular constructs that can better mimic the native tissues when combined with microfluidic systems. Finally, the ongoing challenges and the future perspectives for the ultimate fabrication of functional constructs for advanced research studies are highlighted.
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http://dx.doi.org/10.1088/1758-5090/aae605DOI Listing
November 2018

A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes.

Sci Rep 2018 09 10;8(1):13532. Epub 2018 Sep 10.

Operational Research Unit, Fondazione di Ricerca e Cura Giovanni Paolo II, Largo Gemelli, Campobasso, 86100, Italy.

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host's vasculature.
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http://dx.doi.org/10.1038/s41598-018-31848-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131510PMC
September 2018

Translational Application of Microfluidics and Bioprinting for Stem Cell-Based Cartilage Repair.

Stem Cells Int 2018 20;2018:6594841. Epub 2018 Feb 20.

Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy.

Cartilage defects can impair the most elementary daily activities and, if not properly treated, can lead to the complete loss of articular function. The limitations of standard treatments for cartilage repair have triggered the development of stem cell-based therapies. In this scenario, the development of efficient cell differentiation protocols and the design of proper biomaterial-based supports to deliver cells to the injury site need to be addressed through basic and applied research to fully exploit the potential of stem cells. Here, we discuss the use of microfluidics and bioprinting approaches for the translation of stem cell-based therapy for cartilage repair in clinics. In particular, we will focus on the optimization of hydrogel-based materials to mimic the articular cartilage triggered by their use as bioinks in 3D bioprinting applications, on the screening of biochemical and biophysical factors through microfluidic devices to enhance stem cell chondrogenesis, and on the use of microfluidic technology to generate implantable constructs with a complex geometry. Finally, we will describe some new bioprinting applications that pave the way to the clinical use of stem cell-based therapies, such as scaffold-free bioprinting and the development of a 3D handheld device for the in situ repair of cartilage defects.
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http://dx.doi.org/10.1155/2018/6594841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838503PMC
February 2018

A phase I, randomized, controlled, dose-ranging study of investigational acellular pertussis (aP) and reduced tetanus-diphtheria-acellular pertussis (TdaP) booster vaccines in adults.

Hum Vaccin Immunother 2018 01 27;14(1):45-58. Epub 2017 Nov 27.

b GSK, Vaccines , Siena , Italy.

Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.
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http://dx.doi.org/10.1080/21645515.2017.1385686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791588PMC
January 2018

PLA short sub-micron fiber reinforcement of 3D bioprinted alginate constructs for cartilage regeneration.

Biofabrication 2017 Nov 14;9(4):044105. Epub 2017 Nov 14.

Warsaw University of Technology, Faculty of Materials Science and Engineering, 02-507 Warsaw, Poland.

In this study, we present an innovative strategy to reinforce 3D-printed hydrogel constructs for cartilage tissue engineering by formulating composite bioinks containing alginate and short sub-micron polylactide (PLA) fibers. We demonstrate that Young's modulus obtained for pristine alginate constructs (6.9 ± 1.7 kPa) can be increased threefold (up to 25.1 ± 3.8 kPa) with the addition of PLA short fibers. Furthermore, to assess the performance of such materials in cartilage tissue engineering, we loaded the bioinks with human chondrocytes and cultured in vitro the bioprinted constructs for up to 14 days. Live/dead assays at day 0, 3, 7 and 14 of in vitro culture showed that human chondrocytes were retained and highly viable (∼80%) within the 3D deposited hydrogel filaments, thus confirming that the fabricated composites materials represent a valid solution for tissue engineering applications. Finally, we show that the embedded chondrocytes during all the in vitro culture maintain a round morphology, a key parameter for a proper deposition of neocartilage extracellular matrix.
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http://dx.doi.org/10.1088/1758-5090/aa90d7DOI Listing
November 2017

Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs.

Methods Mol Biol 2017 ;1612:369-380

Department of Chemistry, University of Rome "La Sapienza", P.le A. Moro 5, 00185, Rome, Italy.

3D bioprinting is an emerging field that can be described as a robotic additive biofabrication technology that has the potential to build tissues or organs. In general, bioprinting uses a computer-controlled printing device to accurately deposit cells and biomaterials into precise architectures with the goal of creating on demand organized multicellular tissue structures and eventually intra-organ vascular networks. The latter, in turn, will promote the host integration of the engineered tissue/organ in situ once implanted. Existing biofabrication techniques still lay behind this goal. Here, we describe a novel microfluidic printing head-integrated within a custom 3D bioprinter-that allows for the deposition of multimaterial and/or multicellular within a single scaffold by extruding simultaneously different bioinks or by rapidly switching between one bioink and another. The designed bioprinting method effectively moves toward the direction of creating viable tissues and organs for implantation in clinic and research in lab environments.
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http://dx.doi.org/10.1007/978-1-4939-7021-6_26DOI Listing
March 2018

Naturally derived proteins and glycosaminoglycan scaffolds for tissue engineering applications.

Mater Sci Eng C Mater Biol Appl 2017 Sep 5;78:1277-1299. Epub 2017 Apr 5.

Warsaw University of Technology, Faculty of Material Science and Engineering, 141 Woloska str., 02-507 Warsaw, Poland. Electronic address:

Tissue engineering (TE) aims to mimic the complex environment where organogenesis takes place using advanced materials to recapitulate the tissue niche. Cells, three-dimensional scaffolds and signaling factors are the three main and essential components of TE. Over the years, materials and processes have become more and more sophisticated, allowing researchers to precisely tailor the final chemical, mechanical, structural and biological features of the designed scaffolds. In this review, we will pose the attention on two specific classes of naturally derived polymers: fibrous proteins and glycosaminoglycans (GAGs). These materials hold great promise for advances in the field of regenerative medicine as i) they generally undergo a fast remodeling in vivo favoring neovascularization and functional cells organization and ii) they elicit a negligible immune reaction preventing severe inflammatory response, both representing critical requirements for a successful integration of engineered scaffolds with the host tissue. We will discuss the recent achievements attained in the field of regenerative medicine by using proteins and GAGs, their merits and disadvantages and the ongoing challenges to move the current concepts to practical clinical application.
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http://dx.doi.org/10.1016/j.msec.2017.04.016DOI Listing
September 2017

Combination of biochemical and mechanical cues for tendon tissue engineering.

J Cell Mol Med 2017 Nov 4;21(11):2711-2719. Epub 2017 May 4.

Department of Biology, Tor Vergata Rome University, Rome, Italy.

Tendinopathies negatively affect the life quality of millions of people in occupational and athletic settings, as well as the general population. Tendon healing is a slow process, often with insufficient results to restore complete endurance and functionality of the tissue. Tissue engineering, using tendon progenitors, artificial matrices and bioreactors for mechanical stimulation, could be an important approach for treating rips, fraying and tissue rupture. In our work, C3H10T1/2 murine fibroblast cell line was exposed to a combination of stimuli: a biochemical stimulus provided by Transforming Growth Factor Beta (TGF-β) and Ascorbic Acid (AA); a three-dimensional environment represented by PEGylated-Fibrinogen (PEG-Fibrinogen) biomimetic matrix; and a mechanical induction exploiting a custom bioreactor applying uniaxial stretching. In vitro analyses by immunofluorescence and mechanical testing revealed that the proposed combined approach favours the organization of a three-dimensional tissue-like structure promoting a remarkable arrangement of the cells and the neo-extracellular matrix, reflecting into enhanced mechanical strength. The proposed method represents a novel approach for tendon tissue engineering, demonstrating how the combined effect of biochemical and mechanical stimuli ameliorates biological and mechanical properties of the artificial tissue compared to those obtained with single inducement.
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http://dx.doi.org/10.1111/jcmm.13186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661263PMC
November 2017
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