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Tissue engineering research has undergone to a revolutionary improvement, thanks to technological advancements, such as the introduction of bioprinting technologies. The ability to develop suitable customized biomaterial inks/bioinks, with excellent printability and ability to promote cell proliferation and function, has a deep impact on such improvements. In this context, printing inks based on chitosan and its derivatives have been instrumental. Thus, the current review aims at providing a comprehensive overview on chitosan-based materials as suitable inks for 3D/4D (bio)printing and their applicability in creating advanced drug delivery platforms and tissue engineered constructs. Furthermore, relevant strategies to improve the mechanical and biological performances of this biomaterial are also highlighted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

One of the main challenges of the interface-tissue engineering is the regeneration of diseased or damaged interfacial native tissues that are heterogeneous both in composition and in structure. In order to achieve this objective, innovative fabrication techniques have to be investigated. This work describes the design, fabrication, and validation of a novel mixing system to be integrated into a double-extruder bioprinter, based on an ultrasonic probe included into a mixing chamber. To validate the quality and the influence of mixing time, different nanohydroxyapatite-gelatin samples were printed. Mechanical characterization, micro-computed tomography, and thermogravimetric analysis were carried out. Samples obtained from three-dimensional bioprinting using the mixing chamber were compared to samples obtained by deposition of the same final solution obtained by manually operated ultrasound probe, showing no statistical differences. Results obtained from samples characterization allow to consider the proposed mixing system as a promising tool for the fabrication of graduated structures which are increasingly being used in interface-tissue engineering.

Skeletal muscle engineering aims at tissue reconstruction to replace muscle loss following traumatic injury or in congenital muscle defects. Skeletal muscle can be engineered by using biodegradable and biocompatible scaffolds that favor myogenic cell adhesion and subsequent tissue organization. In this study, we characterized scaffolds made of gelatin cross-linked with genipin, a natural derived cross-linking agent with low cytotoxicity and high biocompatibility, for tissue engineering of skeletal muscle. We generated gelatin-genipin hydrogels with a stiffness of 13 kPa to reproduce the mechanical properties characteristic of skeletal muscle and we show that their surface can be topographically patterned through soft lithography to drive myogenic cells differentiation and unidirectional orientation. Furthermore, we demonstrate that these biomaterials can be successfully implanted in vivo under dorsal mouse skin, showing good biocompatibility and slow biodegradation rate. Moreover, the grafting of this biomaterial in partially ablated tibialis anterior muscle does not impair muscle regeneration, supporting future applications of gelatin-genipin biomaterials in the field of skeletal muscle tissue repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2763-2777, 2018., (© 2018 Wiley Periodicals, Inc.)

The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment is a challenge for regenerative medicine. The creation of sites for the selective binding of specific endogenous proteins represents an attractive strategy to fabricate scaffolds able to elicit specific cell response. Here, electrospinning (ESP) and soft-molecular imprinting (soft-MI) techniques were combined to fabricate a soft-molecular imprinted electrospun bioactive scaffold (SMIES) for tissue regeneration. Scaffolds functionalized using different proteins and growth factors (GFs) arranged onto the surface were designed, fabricated and validated with different polyesters, demonstrating the versatility of the developed approach. The scaffolds bound selectively each of the different proteins used, indicating that the soft-MI method allowed fabricating high affinity binding sites on ESP fibers compared to non-imprinted ones. The imprinting of ESP fibers with several GFs resulted in a significant effect on cell behavior. FGF-2 imprinted SMIES promoted cell proliferation and metabolic activity. BMP-2 and TGF-β3 imprinted SMIES promoted cellular differentiation. These scaffolds hold the potential to be used in a cell-free approach to steer endogenous tissue regeneration in several regenerative medicine applications.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Genipin is a natural low-toxic cross-linker for molecules with primary amino groups, and its use with collagen and gelatin has shown a great potential in tissue engineering applications. The fabrication of scaffolds with a well-organized micro and macro topology using additive manufacturing systems requires an accurate control of working parameters, such as reaction rate, gelling time, and diffusion constant. A polymeric system of 5% w/v gelatin in PBS with 2 mg/mL collagen solutions in a 1:1 weight ratio was used as template to perform measurements varying genipin concentration in a range of 0.1-1.5% w/w with respect to gelatin. In the first part of this work, the reaction rate of the polymeric system was estimated using a new colorimetric analysis of the reaction. Then its workability time, closely related to the gelling time, was evaluated thanks to rheological analysis: finally, the quantification of static and dynamic diffusion constants of genipin across nonreacting and reacting membranes, made respectively by agarose and gelatin, was performed. It was shown that the colorimetric analysis is a good indicator of the reaction progress. The gelling time depends on the genipin concentration, but a workability window of 40 min guaranteed up to 0.5% w/w genipin. The dynamic diffusion constant of genipin in the proposed polymeric system is in the order of magnitude of 10 -7 . The obtained results indicated the possibility to use the genipin, gelatin, and collagen, in the proposed concentrations, to build well-defined hydrogel scaffolds with both extrusion-based and 3D ink-jet system. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 473-480, 2017., (© 2015 Wiley Periodicals, Inc.)

The development of new human skeletal muscle tissue is an alternative approach to the replacement of tissue after severe damage, for example in the case of traumatic injury, where surgical reconstruction is often needed following major loss of natural tissue. Treatment to date has involved the transfer of muscle tissue from other sites, resulting in a functional loss and volume deficiency of donor sites. Approaches that seek to eliminate these problems include the relatively new solution of skeletal muscle engineering. Here there are two main components to consider: (a) the cells with their regenerative potential; and (b) the polymeric structure onto which cells are seeded and where they must perform their activities. In this paper we describe well-defined two- and three-dimensional polymeric structures able to drive the myoblast process of adhesion, proliferation and differentiation. We examine a series of polymers and protein adhesions with which to functionalize the structures, and cell-seeding methods, with a view to defining the optimal protocol for engineering skeletal muscle tissue. All polymer samples were tested for their mechanical and biological properties, to support the validity of our results in the real context of muscle tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd., (Copyright © 2014 John Wiley & Sons, Ltd.)

In this article, conductive hollow fibers have been fabricated using melt spinning technique. Multiwalled carbon nanotubes (MWNTs) and poly(3-hexylthiophene-2,5-diyl) (P3HT) have been used to fabricate conductive poly-caprolactone (PCL) composites. The hollow fibers have inner and outer diameter in the range of 300 µm and 500 µm, respectively. Critical parameters to tune the dimension of hollow fibers have been defined following two-dimensions mathematical model. Evaluation of the mechanical properties showed that the incorporation of 1-3 wt % MWNTs and 5-8 wt % P3HT increased Young Modulus of 10% and 20% respectively, compared with pure PCL. The electrical property assessment demonstrated that a minimum incorporation of 3 wt % MWNT and 8 wt % P3HT in PCL matrix transformed composite materials into conductive materials. In addition, SH-SY5Y human neuroblastoma cells were seeded on the fabricated samples an their adhesion, proliferation and neurite length growth were analysed. In particular we observed that these materials promoted cell activities and in particular on MWNT/PCL composites there was a significant increase of neurite growth., (© 2014 Wiley Periodicals, Inc.)

Chronic wounds such as diabetic foot ulcers and venous leg ulcers are common problems in people suffering from type 2 diabetes. These can cause pain, and nerve damage, eventually leading to foot or leg amputation. These types of wounds are very difficult to treat and sometimes take months or even years to heal because of many possible complications during the process. Allogeneic skin grafting has been used to improve wound healing, but the majority of grafts do not survive several days after being implanted. We have been studying the behavior of fibroblasts and keratinocytes in engineered capillary-like endothelial networks. A dermo-epidermal graft has been implanted in an athymic nude mouse model to assess the integration with the host tissue as well as the wound healing process. To build these networks into a skin graft, a modified inkjet printer was used, which allowed the deposit of human microvascular endothelial cells. Neonatal human dermal fibroblast cells and neonatal human epidermal keratinocytes were manually mixed in the collagen matrix while endothelial cells printed. A full-thickness wound was created at the top of the back of athymic nude mice and the area was covered by the bilayered graft. Mice of the different groups were followed until completion of the specified experimental time line, at which time the animals were humanely euthanized and tissue samples were collected. Wound contraction improved by up to 10% when compared with the control groups. Histological analysis showed the neoskin having similar appearance to the normal skin. Both layers, dermis and epidermis, were present with thicknesses resembling normal skin. Immunohistochemistry analysis showed favorable results proving survival of the implanted cells, and confocal images showed the human cells' location in the samples that were collocated with the bilayer printed skin graft.

The adaptation of inkjet printing technology for the realisation of controlled micro- and nano-scaled biological structures is of great potential in tissue and biomaterial engineering. In this paper we present the Olivetti BioJet system and its applications in tissue engineering and cell printing. BioJet, which employs a thermal inkjet cartridge, was used to print biomolecules and living cells. It is well known that high stresses and forces are developed during the inkjet printing process. When printing living particles (i.e., cell suspensions) the mechanical loading profile can dramatically damage the processed cells. Therefore computational models were developed to predict the velocity profile and the mechanical load acting on a droplet during the printing process. The model was used to investigate the role of the stiffness of the deposition substrate during droplet impact and compared with experimental investigations on cell viability after printing on different materials. The computational model and the experimental results confirm that impact forces are highly dependent on the deposition substrate and that soft and viscous surfaces can reduce the forces acting on the droplet, preventing cell damage. These results have high relevance for cell bioprinting; substrates should be designed to have a good compromise between substrate stiffness to conserve spatial patterning without droplet coalescence but soft enough to absorb the kinetic energy of droplets in order to maintain cell viability., (Copyright © 2011. Published by Elsevier B.V.)

A novel squeeze pressure bioreactor for noncontact hydrodynamic stimulation of cartilage is described. The bioreactor is based on a small piston that moves up and down, perpendicular to a tissue construct, in a fluid-filled chamber. Fluid displaced by the piston generates a pressure wave and shear stress as it moves across the sample, simulating the dynamic environment of a mobile joint. The fluid dynamics inside the squeeze pressure bioreactor was modeled using analytical and computational methods to simulate the mechanical stimuli imposed on a construct. In particular, the pressure, velocity field, and wall shear stress generated on the surface of the construct were analyzed using the theory of hydrodynamic lubrication, which describes the flow of an incompressible fluid between two surfaces in relative motion. Both the models and in-situ pressure measurements in the bioreactor demonstrate that controlled cyclic stresses of up to 10 kPa can be applied to tissue constructs. Initial tests on three-dimensional scaffolds seeded with chondrocytes show that glycosaminoglycan production is increased with regard to controls after 24 and 48 h of cyclic noncontact stimulation in the bioreactor.

The evaluation of viscoelastic properties of human medial patello-femoral ligament is fundamental to understand its physiological function and contribution as stabilizer for the selection of the methods of repair and reconstruction and for the development of scaffolds with adequate mechanical properties. In this work, 12 human specimens were tested to evaluate the time- and history-dependent non linear viscoelastic properties of human medial patello-femoral ligament using the quasi-linear viscoelastic (QLV) theory formulated by Fung et al. (1972) and modified by Abramowitch and Woo (2004) . The five constant of the QLV theory, used to describe the instantaneous elastic response and the reduced relaxation function on stress relaxation experiments, were successfully evaluated. It was found that the constant A was 1.21±0.96 MPa and the dimensionless constant B was 26.03±4.16. The magnitude of viscous response, the constant C , was 0.11±0.02 and the initial and late relaxation time constants τ 1 and τ 2 were 6.32±1.76 s and 903.47±504.73 s respectively. The total stress relaxation was 32.7±4.7%. To validate our results, the obtained constants were used to evaluate peak stresses from a cyclic stress relaxation test on three different specimens. The theoretically predicted values fit the experimental ones demonstrating that the QLV theory could be used to evaluate the viscoelastic properties of the human medial patello-femoral ligament. [ABSTRACT FROM AUTHOR]

Purpose -- This paper aims to develop a novel micro-ablation system to realise micrometric and well-defined hydrogel structures. To engineer a tissue it is necessary to evaluate several aspects, such as cell-cell and cell-substrate interactions, its micro-architecture and mechanical stimuli that act on it. For this reason, it is important to fabricate a substrate which presents a microtopology similar to natural tissue and has chemical and mechanical properties able to promote cell functions. In this paper, well-defined hydrogel structures embedding cells were microfabricated using a purposely developed technique, micro-laser ablation, based on a thulium laser. Its working parameters (laser power emission, stepper motor velocity) were optimised to produce shaded "serpentine" pattern on a hydrogel film. Design/methodology/approach -- In this study, initially, swelling/contraction tests on agarose and alginate hydrogel in different solutions of main components of cell culture medium were performed and were compared with the MECpH model. This comparison matched with good approximation experimental measurements. Once known how hydrogel changed its topology, microstructures with a well-defined topology were realised using a purposely developed micro-laser ablation system design. S5Y5 neuroblastoma cell lines were embedded in hydrogel matrix and the whole structure was ablated with a laser microfabrication system. The cells did not show damages due to mechanical stress present in the hydrogel matrix and to thermal increase induced by the laser beam. Findings -- The hydrogel structure is able to reproduce extracellular matrix. Initially, the hydrogel swelling/contraction in different solutions, containing the main components of the most common cell culture media, was analysed. This analysis is important to evaluate if cell culture environment could alter microtopology of realised structures. Then, the same topology was realised on hydrogel film embedding neuronal cells and the cells did not show damages due to mechanical stress present in the hydrogel matrix and to thermal increase induced by the laser beam. The interesting obtained results could be useful to realise well-defined microfabricated hydrogel structures embedding cells to guide tissue formation Originality/value -- The originality of this paper is the design and realisation of a 3D microfabrication system able to microfabricate hydrogel matrix embedding cells without inducing cell damage. The ease of use of this system and its potential modularity render this system a novel potential device for application in tissue engineering and regenerative medicine area. [ABSTRACT FROM AUTHOR]