3D bioprinting of miniaturized tissues embedded in self-assembled nanoparticle-based fibrillar platforms

The creation of microphysiological systems like tissue and organ-on-chip for in vitro modeling of human physiology and diseases is gathering increasing interest. However, the platforms used to build these systems have limitations concerning implementation, automation, and cost-effectiveness. Moreover, their typical plastic-based housing materials are poor recreations of native tissue extracellular matrix (ECM) and barriers. Here, the controlled self-assembly of plant-derived cellulose nanocrystals (CNC) is combined with the concept of 3D bioprinting in suspension baths for the direct biofabrication of microphysiological systems embedded within an ECM mimetic fibrillar support material. The developed support CNC fluid gel allows exceptionally high-resolution bioprinting of 3D constructs with arbitrary geometries and low restrictions of bioink choice. The further induction of CNC self-assembly with biocompatible calcium ions results in a transparent biomimetic nanoscaled fibrillar matrix that allows hosting different compartmentalized cell types and perfusable channels, has tailored permeability for biomacromolecules diffusion and cellular crosstalk, and holds structural stability to support long-term in vitro cell maturation. In summary, this xeno-free nanoscale CNC fibrillar matrix allows the biofabrication of hierarchical living constructs, opening new opportunities not only for developing physiologically relevant 3D in vitro models but also for a wide range of applications in regenerative medicine.The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples and Hospital Sao Joao (Porto, Portugal) for providing platelet concentrates. The authors acknowledge the financial support from project NORTE-01-0145-FEDER-000021 supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); the European Union Framework Program for Research and Innovation HORIZON 2020, under the Twinning grant agreement no. 810850-Achilles, European Research Council grant agreement no. 772817, Fundacao para a Ciencia e a Tecnologia for the PhD grant for S.M.B PD/BD/129403/2017 financed through doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC), and project PTDC/NAN-MAT/30595/2017. Schematics in Figures 1, 2, and 6 were created with BioRender.com. The authors thank Milan Sixt and Barbara B. Mendes for preliminary tests with CNC fluid gel. The authors thank David Caballero, Catarina Abreu, and Mandana Mombeinipour for providing endothelial cells and Virginia Brancato for breast cancer cells

Blood derivatives awaken in regenerative medicine strategies to modulate wound healing

Blood components play key roles in the modulation of the wound healing process and, together with the provisional fibrin matrix ability to selectively bind bioactive molecules and control its spatial-temporal presentation, define the complex microenvironment that characterize this biological process. As a biomimetic approach, the use of blood derivatives in regenerative strategies has awakened as a source of multiple therapeutic biomolecules. Nevertheless, and despite their clinical relevance, blood derivatives have been showing inconsistent therapeutic results due to several factors, including proper control over their delivery mechanisms. Herein, we highlight recent trends on the use biomaterials to protect, sequester and deliver these pools of biomolecules in tissue engineering and regenerative medicine approaches. Particular emphasis is given to strategies that enable to control their spatiotemporal delivery and improve the selectivity of presentation profiles of the biomolecules derived from blood derivatives rich in platelets. Finally, we discussed possible directions for biomaterials design to potentiate the aimed regenerative effects of blood derivatives and achieve efficient therapies.BBM acknowledges the financial support from FCT/MCTES (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia, e Ensino Superior) and the Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH), PD/59/2013 for PD/BD/113807/2015. MGF acknowledges European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 706996 (PrinTendon). PB acknowledges RECOGNIZE and NORTE2020 (UTAP-ICDT/CTM-BIO/0023/2014). RMD acknowledges SFRH/BPD/112459/2015.info:eu-repo/semantics/publishedVersio

Guiding stem cell tenogenesis by modulation of growth factor signaling and cell-scale biophysical cues in bioengineered constructs

Tendon injuries and tendinopathies are increasingly prevalent health problems currently lacking effective treatments. Tissue engineering offers promising strategies to boost the low innate regenerative ability of tendons. Within this context, the simultaneous leveraging of both physical and biochemical cues by engineered scaffolding systems can be explored to promote a stronger tenogenic response from stem cells. Here, molecularly imprinted polymeric nanoparticles (MINPs) against transforming growth factor (TGF)-β3 are combined with bioinspired anisotropic hydrogels to produce tenogenesis-inductive constructs. MINPs are first solid phase-imprinted against a TGF-β3 epitope, achieving an affinity comparable to monoclonal antibodies. MINPs and magnetically-responsive microfibers are then encapsulated together with adipose-derived stem cells within gelatin-based hydrogels, applying a magnetostatic field during gelation to align the microfibers. The created anisotropic microstructure guides cell growth and elongation unidirectionally, while MINPs act as artificial receptors for TGF-β3, potentiating its paracrine action in the cellular microenvironment. The combination of both stimuli proves effective at increasing TGF-β signaling, which promotes the expression of tendon-associated genes and corresponding protein synthesis, suggesting that microstructural cues and biomolecule sequestration act in tandem to direct cell fate commitment. Overall, this system recapitulates several elements of tendon development, constituting a promising strategy for the regeneration of this tissue

Human-based fibrillar nanocomposite hydrogels as bioinstructive matrices to tune stem cell behavior

The extracellular matrix (ECM)-biomimetic fibrillar structure of platelet lysate (PL) gels along with its enriched milieu of biomolecules has drawn significant interest in regenerative medicine applications. However, PL-based gels have poor structural stability which severely limits its performance as a bioinstructive biomaterial. Here, rod-shaped cellulose nanocrystals (CNC) are used as a novel approach to modulate the physical and biochemical microenvironment of PL gels enabling their effective use as injectable human-based cell scaffolds with a level of biomimicry that is difficult to recreate with synthetic biomaterials. The incorporation of CNC (0 to 0.61 wt.%) into the PL fibrillar network during the coagulation cascade leads to decreased fiber branching, increased interfiber porosity (from 66 to 83%) and modulate fiber (from 1.4 ± 0.7 to 27 ± 12 kPa) and bulk hydrogel (from 18 ± 4 to 1256 ± 82 Pa) mechanical properties. As result of these physicochemical alterations, nanocomposite PL hydrogels resist to the typical extensive clot retraction (from 76 ± 1 to 24 ± 3 at Day 7) and show favored retention of PL bioactive molecules. The feedback of these cues on the fate of human adipose-derived stem cells is evaluated, showing how it can be explored to modulate the commitment of encapsulated stem cells toward different genetic phenotypes without the need for additional external biological stimuli. These fibrillar nanocomposite hydrogels allow therefore to explore the outstanding biological properties of human-based PL as an efficient engineered ECM which can be tailored to trigger specific regenerative pathways in minimal invasive strategies.The authors thank the Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples. The authors acknowledge the financial support from project Recognize (UTAP-ICDT/CTM-BIO/0023/2014), project NORTE-01-0145FEDER-000021 supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No. 739572 – The Discoveries CTR EU, Forecast 668983, Marie Skłodowska-Curie grant agreement No. 706996 (PrinTendon) and CHEM2NATURE 692333; FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) and the Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH) in the framework of PhD grant PD/59/2013 – PD/BD/113807/2015 for BBM, Post-Doc grant SFRH/BPD/112459/2015 for R.D.info:eu-repo/semantics/publishedVersio

Biphasic hydrogels integrating mineralized and anisotropic features for interfacial tissue engineering

The innate graded structural and compositional profile of musculoskeletal tissue interfaces is disrupted and replaced by fibrotic tissue in the context of disease and degeneration. Tissue engineering strategies focused on the restoration of the transitional complexity found in those junctions present special relevance for regenerative medicine. Herein, we developed a gelatin-based multiphasic hydrogel system, where sections with distinct composition and microstructure were integrated in a single unit. In each phase, hydroxyapatite particles or cellulose nanocrystals (CNC) were incorporated into an enzymatically cross-linked gelatin network to mimic bone or tendon tissue, respectively. Stiffer hydrogels were produced with the incorporation of mineralized particles, and magnetic alignment of CNC resulted in anisotropic structure formation. The evaluation of the biological commitment with human adipose-derived stem cells toward the tendon-to-bone interface revealed an aligned cell growth and higher synthesis and deposition of tenascin in the anisotropic phase, while the activity of the secreted alkaline phosphatase and the expression of osteopontin were induced in the mineralized phase. These results highlight the potential versatility offered by gelatin-transglutaminase enzyme tandem for the development of strategies that mimic the graded, composite, and complex intersections of the connective tissues.The authors acknowledge the European Union’s Horizon 2020 research and innovation program under the Teaming grant agreement No 739572 –The Discoveries CTR and European Research Council grant agreement No 726178 -MagTendon; Fundação para a Ciência e a Tecnologia (FCT) for Post-Doc grant SFRH/BPD/112459/2015 and project SmarTendon (PTDC/NAN-MAT/30595/2017); Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund for NORTE-01-0145-FEDER-000021; Spanish Ministry of Economy, ndustry and Competitiveness for the project SAF2016-76150-R. Hospital da Prelada (Portugal) is as well acknowledged for providing the tissue samples. Echave MC thanks the Basque Government for the fellowship grant

Human platelet lysate-based nanocomposite bioink for bioprinting hierarchical fibrillar structures

Three-dimensional (3D) bioprinting holds the promise to fabricate tissue and organ substitutes for regenerative medicine. However, the lack of bioactive inks to fabricate and support functional living constructs is one of the main limitations hindering the progress of this technology. In this study, a biofunctional human-based nanocomposite bioink (HUink) composed of platelet lysate hydrogels reinforced by cellulose nanocrystals is reported. When combined with suspended bioprinting technologies, HUink allows the biofabrication of 3D freeform constructs with high resolution and integrity, mimicking the hierarchical nano-to-macro fibrillary composition of native tissues. Remarkably, HUink supports bioprinting of stem cells with high viability immediately after extrusion and over long-term cell culture without the need for additional biochemical or animal-derived media supplementation. As opposed to typical polymer-based bioinks, the pool of growth factors, cytokines and adhesion proteins in HUink boosts cell spreading and proliferation, stimulating the fast production of cell-secreted extracellular matrix. This innovative bioprinting platform with unpaired biofunctionality allows the fabrication of complex freeform cell-laden constructs that can ultimately be applied in the development of xeno-free 3D tissue models for in vitro research or to develop tissue and organ surrogates for clinical applications.The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples and Hospital São João (Porto, Portugal) for providing platelet concentrates. Authors acknowledge the financial support from project NORTE-010145-FEDER-000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR EU, Marie Skłodowska-Curie grant agreement No 706996 and European Research Council grant agreement No 726178; FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) and the Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH) in the framework of PhD grant PD/59/2013 - PD/BD/113807/2015 for BBM, Post-Doc grant SFRH/BPD/112459/2015 for RMD. The authors would like to thank Tram Le, Carlos Guimarães and Luca Gasperini for their support to acquire pictures and design the drawings. The authors would also like to thank Syeda Mahwish Bakht for the gelatin rhelogical data acquisiton

Refining geomagnetic field intensity changes in Europe between 200 CE and 1800 CE. New data from the Mediterranean region

Absolute past geomagnetic intensity values can mainly be recovered by fired archaeological materials and volcanic rocks. Here, we present 10 new archeointensities from the Mediterranean region that help to better constrain geomagnetic field intensity changes in Europe over the last two millennia. The new archeointensity results were obtained from the Thellier classical method including thermoremanent magnetization (pTRM) checks and both the TRM anisotropy and cooling rate corrections and were derived from at least three specimens. The new data presented, together with a selection of previous archeointensities satisfying a set of quality criteria, confirm the presence of several intensity maxima in Europe over the last 2000 years. In particular, the new archeointensities allow to better define the starting point of the double-oscillation feature that occurred in Europe during the second half of the first millennium CE, and reinforce the existence of a relative maximum at the end of the 14th century - beginning of the 15th century in Western Europe. From selected European archeointensities two new paleosecular variation curves are constructed for Western and Eastern Europe using temporal cubic b-splines in a bootstrap approach. The obtained curves suggest that the occurrence of the intensity maxima is characterized by a period of about 300 ± 50 years. In addition, our results suggest that the maxima do not occur simultaneously in Western and Eastern Europe, pointing out an intensity eastward drift with a mean lag-time of about 100 years