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Article: Editorial: Mechanical behavior of hydrogels for soft tissue replacement and regeneration.

Todros, Silvia / Castilho, Miguel / Espino, Daniel M / Pavan, Piero G

Frontiers in bioengineering and biotechnology

2023 Volume 11, Page(s) 1254076

Language	English
Publishing date	2023-07-25
Publishing country	Switzerland
Document type	Editorial
ZDB-ID	2719493-0
ISSN	2296-4185
ISSN	2296-4185
DOI	10.3389/fbioe.2023.1254076
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Injectable hydrogels for cartilage and bone tissue regeneration: A review

Ghandforoushan, Parisa / Alehosseini, Morteza / Golafshan, Nasim / Castilho, Miguel / Dolatshahi-Pirouz, Alireza / Hanaee, Jalal / Davaran, Soodabeh / Orive, Gorka

International Journal of Biological Macromolecules. 2023 Aug., v. 246 p.125674-

2023

Abstract: Annually, millions of patients suffer from irreversible injury owing to the loss or failure of an organ or tissue caused by accident, aging, or disease. The combination of injectable hydrogels and the science of stem cells have emerged to address this ... ...

Abstract	Annually, millions of patients suffer from irreversible injury owing to the loss or failure of an organ or tissue caused by accident, aging, or disease. The combination of injectable hydrogels and the science of stem cells have emerged to address this persistent issue in society by generating minimally invasive treatments to augment tissue function. Hydrogels are composed of a cross-linked network of polymers that exhibit a high-water retention capacity, thereby mimicking the wet environment of native cells. Due to their inherent mechanical softness, hydrogels can be used as needle-injectable stem cell carrier materials to mend tissue defects. Hydrogels are made of different natural or synthetic polymers, displaying a broad portfolio of eligible properties, which include biocompatibility, low cytotoxicity, shear-thinning properties as well as tunable biological and physicochemical properties. Presently, novel ongoing developments and native-like hydrogels are increasingly being used broadly to improve the quality of life of those with disabling tissue-related diseases. The present review outlines various future and in-vitro applications of injectable hydrogel-based biomaterials, focusing on the newest ongoing developments of in-situ forming injectable hydrogels for bone and cartilage tissue engineering purposes.
Keywords	accidents ; biocompatibility ; biocompatible materials ; bones ; cartilage ; crosslinking ; cytotoxicity ; hardness ; hydrogels ; quality of life ; society ; stem cells ; tissue repair ; wet environmental conditions ; Biomaterials ; Bioactive scaffolds ; Injectable ; Tissue engineering ; Medical applications
Language	English
Dates of publication	2023-08
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	282732-3
ISSN	1879-0003 ; 0141-8130
ISSN (online)	1879-0003
ISSN	0141-8130
DOI	10.1016/j.ijbiomac.2023.125674
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Unveiling the potential of melt electrowriting in regenerative dental medicine.

Daghrery, Arwa / de Souza Araújo, Isaac J / Castilho, Miguel / Malda, Jos / Bottino, Marco C

Acta biomaterialia

2022 Volume 156, Page(s) 88–109

Abstract: For nearly three decades, tissue engineering strategies have been leveraged to devise effective therapeutics for dental, oral, and craniofacial (DOC) regenerative medicine and treat permanent deformities caused by many debilitating health conditions. In ... ...

Abstract	For nearly three decades, tissue engineering strategies have been leveraged to devise effective therapeutics for dental, oral, and craniofacial (DOC) regenerative medicine and treat permanent deformities caused by many debilitating health conditions. In this regard, additive manufacturing (AM) allows the fabrication of personalized scaffolds that have the potential to recapitulate native tissue morphology and biomechanics through the utilization of several 3D printing techniques. Among these, melt electrowriting (MEW) is a versatile direct electrowriting process that permits the development of well-organized fibrous constructs with fiber resolutions ranging from micron to nanoscale. Indeed, MEW offers great prospects for the fabrication of scaffolds mimicking tissue specificity, healthy and pathophysiological microenvironments, personalized multi-scale transitions, and functional interfaces for tissue regeneration in medicine and dentistry. Excitingly, recent work has demonstrated the potential of converging MEW with other AM technologies and/or cell-laden scaffold fabrication (bioprinting) as a favorable route to overcome some of the limitations of MEW for DOC tissue regeneration. In particular, such convergency fabrication strategy has opened great promise in terms of supporting multi-tissue compartmentalization and predetermined cell commitment. In this review, we offer a critical appraisal on the latest advances in MEW and its convergence with other biofabrication technologies for DOC tissue regeneration. We first present the engineering principles of MEW and the most relevant design aspects for transition from flat to more anatomically relevant 3D structures while printing highly-ordered constructs. Secondly, we provide a thorough assessment of contemporary achievements using MEW scaffolds to study and guide soft and hard tissue regeneration, and draw a parallel on how to extrapolate proven concepts for applications in DOC tissue regeneration. Finally, we offer a combined engineering/clinical perspective on the fabrication of hierarchically organized MEW scaffold architectures and the future translational potential of site-specific, single-step scaffold fabrication to address tissue and tissue interfaces in dental, oral, and craniofacial regenerative medicine. STATEMENT OF SIGNIFICANCE: Melt electrowriting (MEW) techniques can further replicate the complexity of native tissues and could be the foundation for novel personalized (defect-specific) and tissue-specific clinical approaches in regenerative dental medicine. This work presents a unique perspective on how MEW has been translated towards the application of highly-ordered personalized multi-scale and functional interfaces for tissue regeneration, targeting the transition from flat to anatomically-relevant three-dimensional structures. Furthermore, we address the value of convergence of biofabrication technologies to overcome the traditional manufacturing limitations provided by multi-tissue complexity. Taken together, this work offers abundant engineering and clinical perspectives on the fabrication of hierarchically MEW architectures aiming towards site-specific implants to address complex tissue damage in regenerative dental medicine.
MeSH term(s)	Regenerative Medicine/methods ; Tissue Scaffolds/chemistry ; Tissue Engineering/methods ; Printing, Three-Dimensional ; Bioprinting/methods
Language	English
Publishing date	2022-01-10
Publishing country	England
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2173841-5
ISSN	1878-7568 ; 1742-7061
ISSN (online)	1878-7568
ISSN	1742-7061
DOI	10.1016/j.actbio.2022.01.010
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Article ; Online: Injectable hydrogels for cartilage and bone tissue regeneration: A review.

Ghandforoushan, Parisa / Alehosseini, Morteza / Golafshan, Nasim / Castilho, Miguel / Dolatshahi-Pirouz, Alireza / Hanaee, Jalal / Davaran, Soodabeh / Orive, Gorka

International journal of biological macromolecules

2023 Volume 246, Page(s) 125674

Abstract	Annually, millions of patients suffer from irreversible injury owing to the loss or failure of an organ or tissue caused by accident, aging, or disease. The combination of injectable hydrogels and the science of stem cells have emerged to address this persistent issue in society by generating minimally invasive treatments to augment tissue function. Hydrogels are composed of a cross-linked network of polymers that exhibit a high-water retention capacity, thereby mimicking the wet environment of native cells. Due to their inherent mechanical softness, hydrogels can be used as needle-injectable stem cell carrier materials to mend tissue defects. Hydrogels are made of different natural or synthetic polymers, displaying a broad portfolio of eligible properties, which include biocompatibility, low cytotoxicity, shear-thinning properties as well as tunable biological and physicochemical properties. Presently, novel ongoing developments and native-like hydrogels are increasingly being used broadly to improve the quality of life of those with disabling tissue-related diseases. The present review outlines various future and in-vitro applications of injectable hydrogel-based biomaterials, focusing on the newest ongoing developments of in-situ forming injectable hydrogels for bone and cartilage tissue engineering purposes.
Language	English
Publishing date	2023-07-04
Publishing country	Netherlands
Document type	Journal Article ; Review
ZDB-ID	282732-3
ISSN	1879-0003 ; 0141-8130
ISSN (online)	1879-0003
ISSN	0141-8130
DOI	10.1016/j.ijbiomac.2023.125674
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Article ; Online: Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration.

Cedillo-Servin, Gerardo / Louro, Ana Filipa / Gamelas, Beatriz / Meliciano, Ana / Zijl, Anne / Alves, Paula M / Malda, Jos / Serra, Margarida / Castilho, Miguel

Biomaterials advances

2023 Volume 155, Page(s) 213692

Abstract: Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin ... ...

Abstract	Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.
MeSH term(s)	Humans ; Induced Pluripotent Stem Cells ; Hydrogels/pharmacology ; Human Umbilical Vein Endothelial Cells ; Extracellular Vesicles ; RNA
Chemical Substances	Hydrogels ; RNA (63231-63-0)
Language	English
Publishing date	2023-11-07
Publishing country	Netherlands
Document type	Journal Article
ISSN	2772-9508
ISSN (online)	2772-9508
DOI	10.1016/j.bioadv.2023.213692
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Article ; Online: Multi-leveled Nanosilicate Implants Can Facilitate Near-Perfect Bone Healing.

Keshavarz, Mozhgan / Alizadeh, Parvin / Kadumudi, Firoz Babu / Orive, Gorka / Gaharwar, Akhilesh K / Castilho, Miguel / Golafshan, Nasim / Dolatshahi-Pirouz, Alireza

ACS applied materials & interfaces

2023 Volume 15, Issue 17, Page(s) 21476–21495

Abstract: Several studies have shown that nanosilicate-reinforced scaffolds are suitable for bone regeneration. However, hydrogels are inherently too soft for load-bearing bone defects of critical sizes, and hard scaffolds typically do not provide a suitable three- ...

Abstract	Several studies have shown that nanosilicate-reinforced scaffolds are suitable for bone regeneration. However, hydrogels are inherently too soft for load-bearing bone defects of critical sizes, and hard scaffolds typically do not provide a suitable three-dimensional (3D) microenvironment for cells to thrive, grow, and differentiate naturally. In this study, we bypass these long-standing challenges by fabricating a cell-free multi-level implant consisting of a porous and hard bone-like framework capable of providing load-bearing support and a softer native-like phase that has been reinforced with nanosilicates. The system was tested with rat bone marrow mesenchymal stem cells in vitro and as a cell-free system in a critical-sized rat bone defect. Overall, our combinatorial and multi-level implant design displayed remarkable osteoconductivity in vitro without differentiation factors, expressing significant levels of osteogenic markers compared to unmodified groups. Moreover, after 8 weeks of implantation, histological and immunohistochemical assays indicated that the cell-free scaffolds enhanced bone repair up to approximately 84% following a near-complete defect healing. Overall, our results suggest that the proposed nanosilicate bioceramic implant could herald a new age in the field of orthopedics.
MeSH term(s)	Rats ; Animals ; Osteogenesis ; Bone and Bones ; Bone Regeneration ; Tissue Scaffolds ; Mesenchymal Stem Cells
Language	English
Publishing date	2023-04-19
Publishing country	United States
Document type	Journal Article
ISSN	1944-8252
ISSN (online)	1944-8252
DOI	10.1021/acsami.3c01717
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Article ; Online: Load-induced fluid pressurisation in hydrogel systems before and after reinforcement by melt-electrowritten fibrous meshes.

Moo, Eng Kuan / Ebrahimi, Mohammadhossein / Hrynevich, Andrei / de Ruijter, Mylène / Castilho, Miguel / Malda, Jos / Korhonen, Rami K

Journal of the mechanical behavior of biomedical materials

2023 Volume 143, Page(s) 105941

Abstract: Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) ... ...

Abstract	Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) technique enables three-dimensional printing of structured fibrous mesh with small fibre diameter (20 μm). The MEW mesh with 20 μm fibre diameter can synergistically increase the instantaneous mechanical stiffness of soft hydrogels. However, the reinforcing mechanism of the MEW meshes is not well understood, and may involve load-induced fluid pressurisation. Here, we examined the reinforcing effect of MEW meshes in three hydrogels: gelatin methacryloyl (GelMA), agarose and alginate, and the role of load-induced fluid pressurisation in the MEW reinforcement. We tested the hydrogels with and without MEW mesh (i.e., hydrogel alone, and MEW-hydrogel composite) using micro-indentation and unconfined compression, and analysed the mechanical data using biphasic Hertz and mixture models. We found that the MEW mesh altered the tension-to-compression modulus ratio differently for hydrogels that are cross-linked differently, which led to a variable change to their load-induced fluid pressurisation. MEW meshes only enhanced the fluid pressurisation for GelMA, but not for agarose or alginate. We speculate that only covalently cross-linked hydrogels (GelMA) can effectively tense the MEW meshes, thereby enhancing the fluid pressure developed during compressive loading. In conclusion, load-induced fluid pressurisation in selected hydrogels was enhanced by MEW fibrous mesh, and may be controlled by MEW mesh of different designs in the future, thereby making fluid pressure a tunable cell growth stimulus for tissue engineering involving mechanical stimulation.
MeSH term(s)	Tissue Scaffolds ; Hydrogels ; Sepharose ; Tissue Engineering/methods ; Gelatin ; Alginates ; Printing, Three-Dimensional
Chemical Substances	Hydrogels ; Sepharose (9012-36-6) ; Gelatin (9000-70-8) ; Alginates
Language	English
Publishing date	2023-05-29
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2378381-3
ISSN	1878-0180 ; 1751-6161
ISSN (online)	1878-0180
ISSN	1751-6161
DOI	10.1016/j.jmbbm.2023.105941
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Article ; Online: GelMA/TCP nanocomposite scaffold for vital pulp therapy.

Han, Yuanyuan / Dal-Fabbro, Renan / Mahmoud, Abdel H / Rahimnejad, Maedeh / Xu, Jinping / Castilho, Miguel / Dissanayaka, Waruna L / Bottino, Marco C

Acta biomaterialia

2023 Volume 173, Page(s) 495–508

Abstract: Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) ... ...

Abstract	Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) fibrous scaffold incorporating beta-tricalcium phosphate (TCP) particles for pulp capping. A comprehensive morphological, physical-chemical, and mechanical characterization of the engineered fibrous scaffolds was performed. In vitro bioactivity, cell compatibility, and odontogenic differentiation potential of the scaffolds in dental pulp stem cells (DPSCs) were also evaluated. A pre-clinical in vivo model was used to determine the therapeutic role of the GelMA/TCP scaffolds in promoting hard tissue formation. Morphological, chemical, and thermal analyses confirmed effective TCP incorporation in the GelMA nanofibers. The GelMA+20%TCP nanofibrous scaffold exhibited bead-free morphology and suitable mechanical and degradation properties. In vitro, GelMA+20%TCP scaffolds supported apatite-like formation, improved cell spreading, and increased deposition of mineralization nodules. Gene expression analysis revealed upregulation of ALPL, RUNX2, COL1A1, and DMP1 in the presence of TCP-laden scaffolds. In vivo, analyses showed mild inflammatory reaction upon scaffolds' contact while supporting mineralized tissue formation. Although the levels of Nestin and DMP1 proteins did not exceed those associated with the clinical reference treatment (i.e., mineral trioxide aggregate), the GelMA+20%TCP scaffold exhibited comparable levels, thus suggesting the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion. Our innovative GelMA/TCP scaffold represents a simplified and efficient alternative to conventional pulp-capping biomaterials. STATEMENT OF SIGNIFICANCE: Vital pulp therapy (VPT) aims to preserve dental pulp vitality and avoid root canal treatment. Biomaterials that bolster mineralized tissue regeneration with ease of use are still lacking. We successfully engineered gelatin methacryloyl (GelMA) electrospun scaffolds incorporated with beta-tricalcium phosphate (TCP) for VPT. Notably, electrospun GelMA-based scaffolds containing 20% (w/v) of TCP exhibited favorable mechanical properties and degradation, cytocompatibility, and mineralization potential indicated by apatite-like structures in vitro and mineralized tissue deposition in vivo, although not surpassing those associated with the standard of care. Collectively, our innovative GelMA/TCP scaffold represents a simplified alternative to conventional pulp capping materials such as MTA and Biodentine™ since it is a ready-to-use biomaterial, requires no setting time, and is therapeutically effective.
MeSH term(s)	Tissue Scaffolds/chemistry ; Cells, Cultured ; Biocompatible Materials/chemistry ; Cell Differentiation ; Apatites/pharmacology ; Dental Pulp
Chemical Substances	beta-tricalcium phosphate ; gelatin methacryloyl ; Biocompatible Materials ; Apatites
Language	English
Publishing date	2023-11-07
Publishing country	England
Document type	Journal Article
ZDB-ID	2173841-5
ISSN	1878-7568 ; 1742-7061
ISSN (online)	1878-7568
ISSN	1742-7061
DOI	10.1016/j.actbio.2023.11.005
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Article ; Online: Experimentally-guided in silico design of engineered heart tissues to improve cardiac electrical function after myocardial infarction.

Rosales, Ricardo M / Mountris, Konstantinos A / Oliván-Viguera, Aida / Pérez-Zabalza, María / Cedillo-Servin, Gerardo / Iglesias-García, Olalla / Hrynevich, Andrei / Castilho, Miguel / Malda, Jos / Prósper, Felipe / Doblaré, Manuel / Mazo, Manuel M / Pueyo, Esther

Computers in biology and medicine

2024 Volume 171, Page(s) 108044

Abstract: Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action ... ...

Abstract	Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm
MeSH term(s)	Humans ; Tissue Engineering/methods ; Induced Pluripotent Stem Cells ; Myocytes, Cardiac/physiology ; Myocardium ; Myocardial Infarction ; Arrhythmias, Cardiac
Language	English
Publishing date	2024-02-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	127557-4
ISSN	1879-0534 ; 0010-4825
ISSN (online)	1879-0534
ISSN	0010-4825
DOI	10.1016/j.compbiomed.2024.108044
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Article ; Online: Covalent Grafting of Functionalized MEW Fibers to Silk Fibroin Hydrogels to Obtain Reinforced Tissue Engineered Constructs.

Viola, Martina / Ainsworth, Madison J / Mihajlovic, Marko / Cedillo-Servin, Gerardo / van Steenbergen, Mies J / van Rijen, Mattie / de Ruijter, Mylène / Castilho, Miguel / Malda, Jos / Vermonden, Tina

Biomacromolecules

2024 Volume 25, Issue 3, Page(s) 1563–1577

Abstract: Hydrogels are ideal materials to encapsulate cells, making them suitable for applications in tissue engineering and regenerative medicine. However, they generally do not possess adequate mechanical strength to functionally replace human tissues, and ... ...

Abstract	Hydrogels are ideal materials to encapsulate cells, making them suitable for applications in tissue engineering and regenerative medicine. However, they generally do not possess adequate mechanical strength to functionally replace human tissues, and therefore they often need to be combined with reinforcing structures. While the interaction at the interface between the hydrogel and reinforcing structure is imperative for mechanical function and subsequent biological performance, this interaction is often overlooked. Melt electrowriting enables the production of reinforcing microscale fibers that can be effectively integrated with hydrogels. Yet, studies on the interaction between these micrometer scale fibers and hydrogels are limited. Here, we explored the influence of covalent interfacial interactions between reinforcing structures and silk fibroin methacryloyl hydrogels (silkMA) on the mechanical properties of the construct and cartilage-specific matrix production
MeSH term(s)	Humans ; Tissue Engineering/methods ; Fibroins/chemistry ; Hydrogels/chemistry ; Cartilage, Articular ; Polymers ; Tissue Scaffolds/chemistry ; Polyesters/chemistry
Chemical Substances	Fibroins (9007-76-5) ; Hydrogels ; Polymers ; Polyesters
Language	English
Publishing date	2024-02-07
Publishing country	United States
Document type	Journal Article
ISSN	1526-4602
ISSN (online)	1526-4602
DOI	10.1021/acs.biomac.3c01147
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