Pectin-Based Scaffolds for Tissue Engineering Applications

Tissue engineering (TE) is an interdisciplinary field that was introduced from the necessity of finding alternative approaches to transplantation for the treatment of damaged and diseased organs or tissues. Unlike the conventional procedures, TE aims at inducing the regeneration of injured tissues through the implantation of customized and functional engineered tissues, built on the so-called ‘scaffolds’. These provide structural support to cells and regulate the process of new tissue formation. The properties of the scaffold are essentials, and they can be controlled by varying the biomaterial formulation and the fabrication technology used to its production. Pectin is emerging as an alternative biomaterial to non-degradable and high-cost petroleum-based biopolymers commonly used in this field. It shows several promising properties including biocompatibility, biodegradability, non-toxicity and gelling capability. Pectin-based formulations can be processed through different fabrication approaches into bidimensional and three-dimensional scaffolds. This chapter aims at highlighting the potentiality in using pectin as biomaterial in the field of tissue engineering. The most representative applications of pectin in preparing scaffolds for wound healing and tissue regeneration are discussed

Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting

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) 3D bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold which underwent osteogenic differentiation for two 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 two 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 four 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

Physicochemical Characterization of Pectin-Gelatin Biomaterial Formulations for 3D Bioprinting.

AbstractDeveloping biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3‐glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion‐based 3D bioprinting. Self‐supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion‐based bioprinting and freeze‐drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue‐like scaffolds for tissue regeneration

Electrospun Structures Made of a Hydrolyzed Keratin-Based Biomaterial for Development of in vitro Tissue Models

The aim of this study is the analysis and characterization of a hydrolyzed keratin-based biomaterial and its processing using electrospinning technology to develop in vitro tissue models. This biomaterial, extracted from poultry feathers, was mixed with type A porcine gelatin and cross-linked with γ-glycidyloxy-propyl-trimethoxy-silane (GPTMS) to be casted initially in the form of film and characterized in terms of swelling, contact angle, mechanical properties, and surface charge density. After these chemical-physical characterizations, electrospun nanofibers structures were manufactured and their mechanical properties were evaluated. Finally, cell response was analyzed by testing the efficacy of keratin-based structures in sustaining cell vitality and proliferation over 4 days of human epithelial, rat neuronal and human primary skin fibroblast cells

Fabrication and characterization of microporous, photocrosslinked poly(trimethylene) carbonate and nano-hydroxyapatite composite. 3D Printing and micro-porosity and roughness effects on the osteogenic differentiation of hBMSCs.

L’obiettivo della tesi è stato quello di sintetizzare un nuovo materiale composito, fotosensibile, biodegradabile e biocompatibile a base d Poli(trimetilene carbonato) (PTMC) e nano-idrossiapatite. Esso è un buon candidato per l’Ingegneria tissutale dell’osso. Usando tre diverse concentrazioni di porogeno (carbonato di etilene) sono stati ottenuti tre diversi inchiostri processabili attraverso la tecnica: low-temperature extrusion-based additive manufacturing (LTEAM) ottenendo strutture tridimensionali. Infine, sono stati studiati gli effetti delle differenti microporosità e rugosità superficiali, dei tre inchiostri, sulla osteogenesi di cellule staminali mesenchimali umane

Green biofabrication of pectin-based scaffolds for Tissue Engineering applications

Reproducing the complex, hierarchical organization and the dynamic functionalities of living tissues remains one of the major challenges in the field of tissue engineering and regenerative medicine. The introduction of bioprinting technologies in these fields played an important role to bridge the gap between natural and engineered tissues. These technologies are a powerful and versatile toolkit that allows to process biomaterials, living cells and bioactive molecules into three-dimensional, tissue-like constructs by additive approaches with unprecedented reproducibility and accuracy. Developing biomaterial formulations that have specific physicochemical properties while being suitable for bioprinting of 3D scaffolds is one of the main bottlenecks limiting the application of bioprinting in clinical scenario. In this thesis, a library of functional bioprintable inks has been successfully developed by combining pectin, a green polysaccharide derived from byproduct of food process industry, and other biopolymers (e.g. gelatin) and nanoparticles (e.g. multiwalled carbon nanotubes). The crosslinking reaction between (3-Glycidyloxypropyl)trimethoxysilane and pectin has been investigated for the first time, and played a key role on obtaining self-supporting, 3D bioprinted scaffolds with complex anatomical shapes (e.g. human ear and nose models). This thesis’s goal is to show that pectin is not only a recycled food thickening agent, but like other biopolymers can be potentially used to produce smart, tailored and multi-tissue implants by extrusion based bioprinting. Therefore, transforming pectin into ecological bioprintable formulations, may open new environmental-friendly perspectives of research, leading to the so-called ‘green biofabrication’ for producing new added-value products

4D Bioprinting as New Tissue Engineering Perspective

Three-dimensional (3D) biological substitutes (scaffolds) able to restore the functions and anatomical properties of living tissues play a key role in Tissue Engineering (TE). Scaffolds can be used to develop patient specific tissue constructs, which once seeded and colonized by the patient cells, can be implanted to induce tissue regeneration avoiding rejection effects. Moreover, they could be used as template for drug screening or to define models of physiological and pathological states. The identification of the chemical and physical properties that should characterize an ideal scaffold is still one of the main challenges of TE. Nonetheless, it is well known that an ideal scaffold has to mimic the natural tissue on the macro- and micro- scale. As the natural extracellular matrix (ECM) surrounding the cells, the scaffold represents the framework for dissociated cells to reform an appropriate tissue structure. The scaffold should reproduce the topological properties (e.g. three-dimensionality, macro- and micro-porosity, pore interconnectivity, surface roughness), mechanical properties (e.g. elastic modulus) and biochemical signalling (e.g. ECM composition) of the living tissue. The more the scaffold is similar to the natural tissue, the higher is the chance that cells recognize it as their natural environment and start to adhere, migrate, proliferate, differentiate and vascularize it. The scaffold features are crucially determined by materials and their biofabrication techniques. Biocompatible and biodegradable polymers (natural or synthetic) and ceramics, with or without inclusion of bioactive molecules, are the main materials used to produce 3D scaffolds. Different fabrication techniques are utilised to obtain these scaffolds, such as solvent casting, freeze drying or bioprinting which include a series of computer aided fabrication techniques.1-2 These last techniques allow to obtain tailored and reproducible scaffolds with a high control over the scaffold architecture, pore geometry and pore interconnectivity by layer-by-layer deposition of biomaterials and living cells. Despite all the advantages, 3D bioprinted scaffolds are not able to reproduce the dynamic activities and functions of living tissues and organs (e.g. heart contractility, gut peristaltic activity and bone remodelling),3 which are very important for the maintenance of the homeostasis. 3D bioprinting considers, in fact, the scaffold as an inanimate and static support neglecting the bidirectional cell-scaffold interactions. Four-dimensional (4D) bioprinting is currently emerging as a promising and innovative biofabrication approach which may have a positive impact and may revolutionize the TE paradigm. Particularly, 4D bioprinting is so called since it introduces the variable ‘time’ as fourth dimension in the 3D bioprinting process. It enables to obtain scaffolds that replicate not only the complex geometry of natural organs but also the ability of living tissues to react to external stimuli at the macro- and micro-scale. This is possible mainly by two different approaches. The first is based on the 3D printing of ‘smart’ materials, namely responsive materials, which are able to reshape or transform themselves in response to external stimuli (e.g. variations of temperature, pH, humidity, electric fields, magnetic fields, light).3 Biocompatible responsive hydrogels are promising material candidates for 4D bioprinting approaches. These materials, in fact, change their shape by swelling or deswelling in response to external stimuli. Furthermore, hydrogels mimic the physical properties of the natural ECM and many of these are printable at physiological temperature (37°C) supporting viable cells during 3D printing process.3-4 One of the most studied hydrogels for 4D bioprinting applications are the thermoresponsive poly(N-isopropylacrylamide)-based polymers that undergo a reversible volume transition at a critical solution temperature close to physiological temperature.5 Natural polymers with cell laden have also been used in 4D bioprinting approach. In this regard, engineered blood vessels were obtained by 3D bioprinting cells encapsulated in alginate and hyaluronic acid on a flat surface. The tube shape was subsequently obtained upon immersion the 3D bioprinted structure in cell culture media. Blood vessels with 20 µm diameter were obtained (which are not yet achievable by other existing biofabrication approaches) and a high cellular alignment on the vessel walls was observed.3-5 The second approach refers to the production of active forces produced upon the maturation of engineered tissue constructs after printing.3 Cell traction forces, originated from intracellular actin polymerization and actomyosin interactions, are used in the method called ‘cell origami’ to produce 3D microstructures by culturing cells on two-dimensional (2D) microplates. The active cellular force causes the folding of the 2D surface in predefined shapes. By changing the geometry of the patterned 2D microplates, various cell-laden structures can be obtained after folding.5 Both these approaches allow to obtain programmable ECM-mimicking scaffolds, namely dynamic constructs whose external stimuli responses are programmed a priori. Particularly, mathematical models provide an important support to simulate and optimize both the scaffold architecture and its composition to obtain predefined functional scaffolds. Compared to 3D scaffolds, 4D scaffolds provides extra stimuli to cell adhesion, proliferation and differentiation to a specific cell phenotype obtaining more realistic engineered tissues. This may have a positive contribution to solve unaddressed worldwide medical needs, such as organ transplantation, by inducing the regeneration of the intended tissue. Finally, thanks to 4D bioprinting it is possible to obtain scaffolds that mimic the complex geometry of organs which can not be obtained by traditional 3D biofabrication techniques to date. Being a new biofabrication approach, 4D bioprinting methods and applications are still currently investigating and several challenges need to be addressed. One of the main challenges of 4D bioprinting is to reproduce the cyclic activity of living organs, such as cardiac contraction, at the same extent and frequency as naturally occurring. Controlling the spatiotemporal response of 4D scaffolds in terms of variation of shape, orientation and/or functionality is in fact arduous. Moreover, the number of printable responsive biomaterials with high sensitivity to specific stimuli at physiological temperatures is limited. Further studies need to be carried out both to synthesize new processable responsive biomaterials and to identify new actuation forms in order to better mimic the functionalities of living tissues. Finally, the effect of the fourth dimension on in vivo tissue regeneration has also to be investigated. In conclusion, 4D bioprinting opens up new perspectives to find interesting and innovative solutions to overcome TE issues

Osteogenic differentiation of hBMSCs on porous photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite composites

Large bone defects are challenging to repair and novel implantable materials are needed to aid in their reconstruction. Research in the past years has proven the beneficial effect of porosity in an implant on osteogenesis in vivo. Building on this research we report here on porous composites based on photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite. These composites were prepared by a temperature induced phase separation of poly(trimethylene carbonate) macromers from solution in ethylene carbonate. By controlling the ethylene carbonate content in viscous dispersions of nano-hydroxyapatite in poly(trimethylene carbonate) macromer solutions, composites with 40 wt% nano-hydroxyapatite and 27 to 71% porosity were prepared. The surface structure of these porous composites was affected by their porosity and their topography became dominated by deep micro-pore channels with the majority of pore widths below 20 µm and rougher surfaces on the nano-scale. The stiffness and toughness of the composites decreased with increasing porosity from 67 to 3.5 MPa and 263 to 2.2 N/mm2, respectively. In cell culture experiments, human bone marrow mesenchymal stem cells proliferated well on the composites irrespective of their porosity. Furthermore, differentiation of the cells was demonstrated by determination of ALP activity and calcium production. The extent of differentiation was affected by the porosity of the films, offering a reduced mechanical incentive for osteogenic differentiation at higher porosities with topographies likely offering a reduced possibility for cells to aggregate and to elongate into morphologies favourable for osteogenic differentiation. This ultimately resulted in a 3-fold reduction of calcium production of the differentiated cells on composites with 71% porosity compared to those on composites with 27% porosity

Pectin-GPTMS-Based Biomaterial: toward a Sustainable Bioprinting of 3D scaffolds for Tissue Engineering Application

Developing green and nontoxic biomaterials, derived from renewable sources and processable through 3D bioprinting technologies, is an emerging challenge of sustainable tissue engineering. Here, pectin from citrus peels was cross-linked for the first time with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) through a one-pot procedure. Freeze-dried porous pectin sponges, with tunable properties in terms of porosity, water uptake, and compressive modulus, were obtained by controlling GPTMS content. Cell experiments showed that GPTMS did not affect the cytocompatibility of pectin. The addition of GPTMS improved the printability of pectin due to an increase of viscosity and yield stress. Three-dimensional woodpile and complex anatomical-shaped scaffolds with interconnected micro- and macropores were, therefore, bioprinted without the use of any additional support material. These results show the great potential of using pectin cross-linked with GPTMS as biomaterial ink to fabricate patient-specific scaffolds, which could be used to promote tissue regeneration in vivo

A novel 3D in vitro model of the human gut microbiota

Clinical trials and animal studies on the gut microbiota are often limited by the difficult access to the gut, restricted possibility of in vivo monitoring, and ethical issues. An easily accessible and monitorable in vitro model of the gut microbiota represents a valid tool for a wider comprehension of the mechanisms by which microbes interact with the host and with each other. Herein, we present a novel and reliable system for culturing the human gut microbiota in vitro. An electrospun gelatin structure was biofabricated as scaffold for microbial growth. The efficiency of this structure in supporting microbial proliferation and biofilm formation was initially assessed for five microbes commonly inhabiting the human gut. The human fecal microbiota was then cultured on the scaffolds and microbial biofilms monitored by confocal laser and scanning electron microscopy and quantified over time. Metagenomic analyses and Real-Time qPCRs were performed to evaluate the stability of the cultured microbiota in terms of qualitative and quantitative composition. Our results reveal the three-dimensionality of the scaffold-adhered microbial consortia that maintain the bacterial biodiversity and richness found in the original sample. These findings demonstrate the validity of the developed electrospun gelatin-based system for in vitro culturing the human gut microbiota