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AUTHOR Dubey, Nileshkumar and Ferreira, Jessica A. and Daghrery, Arwa and Aytac, Zeynep and Malda, Jos and Bhaduri, Sarit B. and Bottino, Marco C.
Title Highly Tunable Bioactive Fiber-Reinforced Hydrogel for Guided Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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One of the most damaging pathologies that affects the health of both soft and hard tissues around the tooth is periodontitis. Clinically, periodontal tissue destruction has been managed by an integrated approach involving elimination of injured tissues followed by regenerative strategies with bone substitutes and/or barrier membranes. Regrettably, a barrier membrane with predictable mechanical integrity and multifunctional therapeutic features has yet to be established. Herein, we report a fiber-reinforced hydrogel with unprecedented tunability in terms of mechanical competence and therapeutic features by integration of highly porous poly(ε-caprolactone) fibrous mesh(es) with well-controlled 3D architecture into bioactive amorphous magnesium phosphate-laden gelatin methacryloyl hydrogels. The presence of amorphous magnesium phosphate and PCL mesh in the hydrogel can control the mechanical properties and improve the osteogenic ability, opening a tremendous opportunity in guided bone regeneration (GBR). Results demonstrate that the presence of PCL meshes fabricated via melt electrowriting can delay hydrogel degradation preventing soft tissue invasion and providing the mechanical barrier to allow time for slower migrating progenitor cells to participate in bone regeneration due to their ability to differentiate into bone-forming cells. Altogether, our approach offers a platform technology for the development of the next-generation of GBR membranes with tunable mechanical and therapeutic properties to amplify bone regeneration in compromised sites.
AUTHOR Daly, Andrew C. and Kelly, Daniel J.
Title Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers [Abstract]
Year 2019
Journal/Proceedings Biomaterials
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Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.
AUTHOR Gonzalez-Fernandez, T. and Rathan, S. and Hobbs, C. and Pitacco, P. and Freeman, F. E. and Cunniffe, G. M. and Dunne, N. J. and McCarthy, H. O. and Nicolosi, V. and O'Brien, F. J. and Kelly, D. J.
Title Pore-forming bioinks to enable Spatio-temporally defined gene delivery in bioprinted tissues [Abstract]
Year 2019
Journal/Proceedings Journal of Controlled Release
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The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.
AUTHOR de Ruijter, Mylène and Ribeiro, Alexandre and Dokter, Inge and Castilho, Miguel and Malda, Jos
Title Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs [Abstract]
Year 2018
Journal/Proceedings Advanced Healthcare Materials
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Abstract Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological tissues.
AUTHOR Cunniffe, Gráinne and Gonzalez-Fernandez, Tomas and Daly, Andrew and Nelson Sathy, Binulal and Jeon, Oju and Alsberg, Eben and J. Kelly, Daniel
Title Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering [Abstract]
Year 2017
Journal/Proceedings Tissue Engineering Part A
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Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-g-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bonemarrow-derived mesenchymal stemcells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization andmineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.
AUTHOR Freeman, Fiona E. and Pitacco, Pierluca and van Dommelen, Lieke H. A. and Nulty, Jessica and Browe, David C. and Shin, Jung-Youn and Alsberg, Eben and Kelly, Daniel J.
Title 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration [Abstract]
Year 2020
Journal/Proceedings Science Advances
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Therapeutic growth factor delivery typically requires supraphysiological dosages, which can cause undesirable off-target effects. The aim of this study was to 3D bioprint implants containing spatiotemporally defined patterns of growth factors optimized for coupled angiogenesis and osteogenesis. Using nanoparticle functionalized bioinks, it was possible to print implants with distinct growth factor patterns and release profiles spanning from days to weeks. The extent of angiogenesis in vivo depended on the spatial presentation of vascular endothelial growth factor (VEGF). Higher levels of vessel invasion were observed in implants containing a spatial gradient of VEGF compared to those homogenously loaded with the same total amount of protein. Printed implants containing a gradient of VEGF, coupled with spatially defined BMP-2 localization and release kinetics, accelerated large bone defect healing with little heterotopic bone formation. This demonstrates the potential of growth factor printing, a putative point of care therapy, for tightly controlled tissue regeneration.
AUTHOR Lee, Ji Seung and Park, Hae Sang and Jung, Harry and Lee, Hanna and Hong, Heesun and Lee, Young Jin and Suh, Ye Ji and Lee, Ok Joo and Kim, Soon Hee and Park, Chan Hum
Title 3D-printable photocurable bioink for cartilage regeneration of tonsil-derived mesenchymal stem cells [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.
AUTHOR Daly, Andrew C. and Pitacco, Pierluca and Nulty, Jessica and Cunniffe, Gráinne M. and Kelly, Daniel J.
Title 3D printed microchannel networks to direct vascularisation during endochondral bone repair [Abstract]
Year 2018
Journal/Proceedings Biomaterials
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Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.
AUTHOR Cao, Chuanliang and Huang, Pengren and Prasopthum, Aruna and Parsons, Andrew J. and Ai, Fanrong and Yang, Jing
Title Characterisation of bone regeneration in 3D printed ductile PCL/PEG/hydroxyapatite scaffolds with high ceramic microparticle concentrations [Abstract]
Year 2022
Journal/Proceedings Biomater. Sci.
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3D printed bioactive glass or bioceramic particle reinforced composite scaffolds for bone tissue engineering currently suffer from low particle concentration (100% breaking strain) by adding poly(ethylene glycol) which is biocompatible and FDA approved. The scaffolds require no post-printing washing to remove hazardous components. More exposure of HA microparticles on strut surfaces is enabled by incorporating higher HA concentrations. Compared to scaffolds with 72 wt% HA{,} scaffolds with higher HA content (90 wt%) enhance matrix formation but not new bone volume after 12 weeks implantation in rat calvarial defects. Histological analyses demonstrate that bone regeneration within the 3D printed scaffolds is via intramembranous ossification and starts in the central region of pores. Fibrous tissue that resembles non-union tissue within bone fractures is formed within pores that do not have new bone. The amount of blood vessels is similar between scaffolds with mainly fibrous tissue and those with more bone tissue{,} suggesting vascularization is not a deciding factor for determining the type of tissues regenerated within the pores of 3D printed scaffolds. Multinucleated immune cells are commonly present in all scaffolds surrounding the struts{,} suggesting a role of managing inflammation in bone regeneration within 3D printed scaffolds.
AUTHOR Yan Li and Lijing Huang and Guangpin Tai and Feifei Yan and Lin Cai and Chenxing Xin and Shamoon {Al Islam}
Title Graphene Oxide-loaded magnetic nanoparticles within 3D hydrogel form High-performance scaffolds for bone regeneration and tumour treatment [Abstract]
Year 2022
Journal/Proceedings Composites Part A: Applied Science and Manufacturing
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The treatment of tumour-related bone defects should ideally combine bone regeneration with tumour treatment. Additive manufacturing (AM) could feasibly place functional bone-repair materials within composite materials with functional-grade structures, giving them bone repair and anti-tumour effects. Magnetothermal therapy is a promising non-invasive method of tumour treatment that has attracted increasing attention. In this study, we prepared novel hydrogel composite scaffolds of polyvinyl alcohol/sodium alginate/hydroxyapatite (PVA/SA/HA) at low temperature via AM. The scaffolds were loaded with various concentrations of magnetic graphene oxide (MGO) @Fe3O4 nanoparticles. The scaffolds were characterised by fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA), which showed that the scaffolds have good moulding qualities and strong hydrogen bonding between the MGO/PVA/SA/HA components. TGA analysis demonstrated the expected thermal stability of the MGO and scaffolds. Thermal effects can be adjusted by varying the contents of MGO and the strength of an external alternating magnetic field. The prepared MGO hydrogel composite scaffolds enhance biological functions and support bone mesenchymal stem cell differentiation in vitro. The scaffolds also show favourable anti-tumour characteristics with effective magnetothermal conversion in vivo.
AUTHOR Zhang, Xiao and Liu, Yang and Zuo, Qiang and Wang, Qingyun and Li, Zuxi and Yan, Kai and Yuan, Tao and Zhang, Yi and Shen, Kai and Xie, Rui and Fan, Weimin
Title 3D Bioprinting of Biomimetic Bilayered Scaffold Consisting of Decellularized Extracellular Matrix and Silk Fibroin for Osteochondral Repair [Abstract]
Year 2021
Journal/Proceedings International Journal of Bioprinting; Vol 7, No 4 (2021)
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Recently, three-dimensional (3D) bioprinting technology is becoming an appealing approach for osteochondral repair. However, it is challenging to develop a bilayered scaffold with anisotropic structural properties to mimic a native osteochondral tissue. Herein, we developed a bioink consisting of decellularized extracellular matrix and silk fibroin to print the bilayered scaffold. The bilayered scaffold mimics the natural osteochondral tissue by controlling the composition, mechanical properties, and growth factor release in each layer of the scaffold. The in vitro results show that each layer of scaffolds had a suitable mechanical strength and degradation rate. Furthermore, the scaffolds encapsulating transforming growth factor-beta (TGF-β) and bone morphogenetic protein-2 (BMP-2) can act as a controlled release system and promote directed differentiation of bone marrow-derived mesenchymal stem cells. Furthermore, the in vivo experiments suggested that the scaffolds loaded with growth factors promoted osteochondral regeneration in the rabbit knee joint model. Consequently, the biomimetic bilayered scaffold loaded with TGF-β and BMP-2 would be a promising strategy for osteochondral repair.
AUTHOR Nulty, Jessica and Freeman, Fiona E. and Browe, David C. and Burdis, Ross and Ahern, Daniel P. and Pitacco, Pierluca and Lee, Yu Bin and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of prevascularised implants for the repair of critically-sized bone defects [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. Statement of Significance This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.
AUTHOR Golafshan, Nasim and Willemsen, Koen and Kadumudi, Firoz Babu and Vorndran, Elke and Dolatshahi-Pirouz, Alireza and Weinans, Harrie and van der Wal, Bart C. H. and Malda, Jos and Castilho, Miguel
Title 3D-Printed Regenerative Magnesium Phosphate Implant Ensures Stability and Restoration of Hip Dysplasia [Abstract]
Year 2021
Journal/Proceedings Advanced Healthcare Materials
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Abstract Osteoarthritis of the hip is a painful and debilitating condition commonly occurring in humans and dogs. One of the main causes that leads to hip osteoarthritis is hip dysplasia. Although the current surgical methods to correct dysplasia work satisfactorily in many circumstances, these are associated with serious complications, tissue resorption, and degeneration. In this study, a one-step fabrication of a regenerative hip implant with a patient-specific design and load-bearing properties is reported. The regenerative hip implant is fabricated based on patient imaging files and by an extrusion assisted 3D printing process using a flexible, bone-inducing biomaterial. The novel implant can be fixed with metallic screws to host bone and can be loaded up to physiological loads without signs of critical permanent deformation or failure. Moreover, after exposing the hip implant to accelerated in vitro degradation, it is confirmed that it is still able to support physiological loads even after losing ≈40% of its initial mass. In addition, the osteopromotive properties of the novel hip implant is demonstrated as shown by an increased expression of osteonectin and osteocalcin by cultured human mesenchymal stem cells after 21 days. Overall, the proposed hip implant provides an innovative regenerative and mechanically stable solution for hip dysplasia treatment.
AUTHOR Chelsea Twohig and Mari Helsinga and Amin Mansoorifar and Avathamsa Athirasala and Anthony Tahayeri and Cristiane Miranda França and Silvia Amaya Pajares and Reyan Abdelmoniem and Susanne Scherrer and Stéphane Durual and Jack Ferracane and Luiz E. Bertassoni
Title A dual-ink 3D printing strategy to engineer pre-vascularized bone scaffolds in-vitro [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.
AUTHOR Bin Wang and Pedro J. Díaz-Payno and David C. Browe and Fiona E. Freeman and Jessica Nulty and Ross Burdis and Daniel J. Kelly
Title Affinity-bound growth factor within sulfated interpenetrate network bioinks for bioprinting cartilaginous tissues [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a ‘single-stage’ or ‘point-of-care’ strategy for regenerating cartilaginous tissues. Statement of Significance: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrate networks in the bioink enables unique high-fidelity bioprinting and unique synergistic mechanical properties. The presence of alginate sulfate provided the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis.
AUTHOR Nulty, Jessica and Burdis, Ross and Kelly, Daniel J.
Title Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.
AUTHOR Falcones, Bryan and Sanz-Fraile, Héctor and Marhuenda, Esther and Mendizábal, Irene and Cabrera-Aguilera, Ignacio and Malandain, Nanthilde and Uriarte, Juan J. and Almendros, Isaac and Navajas, Daniel and Weiss, Daniel J. and Farré, Ramon and Otero, Jorge
Title Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells [Abstract]
Year 2021
Journal/Proceedings Polymers
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Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.
AUTHOR Fenelon, Mathilde and Etchebarne, Marion and Siadous, Robin and Grémare, Agathe and Durand, Marlène and Sentilhes, Loic and Catros, Sylvain and Gindraux, Florelle and L'Heureux, Nicolas and Fricain, Jean-Christophe
Title Comparison of amniotic membrane versus the induced membrane for bone regeneration in long bone segmental defects using calcium phosphate cement loaded with BMP-2 [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.
AUTHOR Petretta, Mauro and Gambardella, Alessandro and Boi, Marco and Berni, Matteo and Cavallo, Carola and Marchiori, Gregorio and Maltarello, Maria Cristina and Bellucci, Devis and Fini, Milena and Baldini, Nicola and Grigolo, Brunella and Cannillo, Valeria
Title Composite Scaffolds for Bone Tissue Regeneration Based on PCL and Mg-Containing Bioactive Glasses [Abstract]
Year 2021
Journal/Proceedings Biology
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Abstract
Polycaprolactone (PCL) is widely used in additive manufacturing for the construction of scaffolds for tissue engineering because of its good bioresorbability, biocompatibility, and processability. Nevertheless, its use is limited by its inadequate mechanical support, slow degradation rate and the lack of bioactivity and ability to induce cell adhesion and, thus, bone tissue regeneration. In this study, we fabricated 3D PCL scaffolds reinforced with a novel Mg-doped bioactive glass (Mg-BG) characterized by good mechanical properties and biological reactivity. An optimization of the printing parameters and scaffold fabrication was performed; furthermore, an extensive microtopography characterization by scanning electron microscopy and atomic force microscopy was carried out. Nano-indentation tests accounted for the mechanical properties of the scaffolds, whereas SBF tests and cytotoxicity tests using human bone-marrow-derived mesenchymal stem cells (BM-MSCs) were performed to evaluate the bioactivity and in vitro viability. Our results showed that a 50/50 wt% of the polymer-to-glass ratio provides scaffolds with a dense and homogeneous distribution of Mg-BG particles at the surface and roughness twice that of pure PCL scaffolds. Compared to pure PCL (hardness H = 35 ± 2 MPa and Young’s elastic modulus E = 0.80 ± 0.05 GPa), the 50/50 wt% formulation showed H = 52 ± 11 MPa and E = 2.0 ± 0.2 GPa, hence, it was close to those of trabecular bone. The high level of biocompatibility, bioactivity, and cell adhesion encourages the use of the composite PCL/Mg-BG scaffolds in promoting cell viability and supporting mechanical loading in the host trabecular bone.
AUTHOR Bello, Thomas and Paindelli, Claudia and Diaz-Gomez, Luis A. and Melchiorri, Anthony and Mikos, Antonios G. and Nelson, Peter S. and Dondossola, Eleonora and Gujral, Taranjit S.
Title Computational modeling identifies multitargeted kinase inhibitors as effective therapies for metastatic, castration-resistant prostate cancer [Abstract]
Year 2021
Journal/Proceedings Proceedings of the National Academy of Sciences
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Abstract
Metastatic, castration-resistant prostate cancer (mCRPC) is an advanced prostate cancer with limited therapeutic options and poor patient outcomes. To investigate whether multitargeted kinase inhibitors (KIs) represent an opportunity for mCRPC drug development, we applied machine learning{textendash}based functional screening and identified two KIs, PP121 and SC-1, which demonstrated strong suppression of CRPC growth in vitro and in vivo. Furthermore, we show the marked ability of these KIs to improve on standard-of-care chemotherapy in both tumor response and survival, suggesting that combining multitargeted KIs with chemotherapy represents a promising avenue for mCRPC treatment. Overall, our findings demonstrate the application of a multidisciplinary strategy that blends bench science with machine-learning approaches for rapidly identifying KIs that result in desired phenotypic effects.Castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer with limited therapeutic options. Here, we applied a systems-based modeling approach called kinome regularization (KiR) to identify multitargeted kinase inhibitors (KIs) that abrogate CRPC growth. Two predicted KIs, PP121 and SC-1, suppressed CRPC growth in two-dimensional in vitro experiments and in vivo subcutaneous xenografts. An ex vivo bone mimetic environment and in vivo tibia xenografts revealed resistance to these KIs in bone. Combining PP121 or SC-1 with docetaxel, standard-of-care chemotherapy for late-stage CRPC, significantly reduced tibia tumor growth in vivo, decreased growth factor signaling, and vastly extended overall survival, compared to either docetaxel monotherapy. These results highlight the utility of computational modeling in forming physiologically relevant predictions and provide evidence for the role of multitargeted KIs as chemosensitizers for late-stage, metastatic CRPC.All study data are included in the article and/or supporting information.
AUTHOR Zhang, Xiao and Liu, Yang and Luo, Chunyang and Zhai, Chenjun and Li, Zuxi and Zhang, Yi and Yuan, Tao and Dong, Shilei and Zhang, Jiyong and Fan, Weimin
Title Crosslinker-free silk/decellularized extracellular matrix porous bioink for 3D bioprinting-based cartilage tissue engineering [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Abstract
As cartilage tissue lacks the innate ability to mount an adequate regeneration response, damage to it is detrimental to the quality of life of the subject. The emergence of three-dimensional bioprinting (3DBP) technology presents an opportunity to repair articular cartilage defects. However, widespread adoption of this technique has been impeded by difficulty in preparing a suitable bioink and the toxicity inherent in the chemical crosslinking process of most bioinks. Our objective was to develop a crosslinker-free bioink with the same biological activity as the original cartilage extracellular matrix (ECM) and good mechanical strength. We prepared bioinks containing different concentrations of silk fibroin and decellularized extracellular matrix (SF-dECM bioinks) mixed with bone marrow mesenchymal stem cells (BMSCs) for 3D bioprinting. SF and dECM interconnect with each other through physical crosslinking and entanglement. A porous structure was formed by removing the polyethylene glycol from the SF-dECM bioink. The results showed the SF-dECM construct had a suitable mechanical strength and degradation rate, and the expression of chondrogenesis-specific genes was found to be higher than that of the SF control construct group. Finally, we confirmed that a SF-dECM construct that was designed to release TGF-β3 had the ability to promote chondrogenic differentiation of BMSCs and provided a good cartilage repair environment, suggesting it is an ideal scaffold for cartilage tissue engineering.
AUTHOR Wang, Weiguang and Chen, Jun-Xiang and Hou, Yanhao and Bartolo, Paulo and Chiang, Wei-Hung
Title Investigations of Graphene and Nitrogen-Doped Graphene Enhanced Polycaprolactone 3D Scaffolds for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Nanomaterials
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Abstract
Scaffolds play a key role in tissue engineering applications. In the case of bone tissue engineering, scaffolds are expected to provide both sufficient mechanical properties to withstand the physiological loads, and appropriate bioactivity to stimulate cell growth. In order to further enhance cell–cell signaling and cell–material interaction, electro-active scaffolds have been developed based on the use of electrically conductive biomaterials or blending electrically conductive fillers to non-conductive biomaterials. Graphene has been widely used as functioning filler for the fabrication of electro-active bone tissue engineering scaffolds, due to its high electrical conductivity and potential to enhance both mechanical and biological properties. Nitrogen-doped graphene, a unique form of graphene-derived nanomaterials, presents significantly higher electrical conductivity than pristine graphene, and better surface hydrophilicity while maintaining a similar mechanical property. This paper investigates the synthesis and use of high-performance nitrogen-doped graphene as a functional filler of poly(ɛ-caprolactone) (PCL) scaffolds enabling to develop the next generation of electro-active scaffolds. Compared to PCL scaffolds and PCL/graphene scaffolds, these novel scaffolds present improved in vitro biological performance.
AUTHOR Trucco, Diego and Sharma, Aarushi and Manferdini, Cristina and Gabusi, Elena and Petretta, Mauro and Desando, Giovanna and Ricotti, Leonardo and Chakraborty, Juhi and Ghosh, Sourabh and Lisignoli, Gina
Title Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting [Abstract]
Year 2021
Journal/Proceedings ACS Biomater. Sci. Eng.
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Abstract
Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications. Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.
AUTHOR Petretta, Mauro and Gambardella, Alessandro and Desando, Giovanna and Cavallo, Carola and Bartolotti, Isabella and Shelyakova, Tatiana and Goranov, Vitaly and Brucale, Marco and Dediu, Valentin Alek and Fini, Milena and Grigolo, Brunella
Title Multifunctional 3D-Printed Magnetic Polycaprolactone/Hydroxyapatite Scaffolds for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Polymers
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Abstract
Multifunctional and resistant 3D structures represent a great promise and a great challenge in bone tissue engineering. This study addresses this problem by employing polycaprolactone (PCL)-based scaffolds added with hydroxyapatite (HAp) and superparamagnetic iron oxide nanoparticles (SPION), able to drive on demand the necessary cells and other bioagents for a high healing efficiency. PCL-HAp-SPION scaffolds with different concentrations of the superparamagnetic component were developed through the 3D-printing technology and the specific topographical features were detected by Atomic Force and Magnetic Force Microscopy (AFM-MFM). AFM-MFM measurements confirmed a homogenous distribution of HAp and SPION throughout the surface. The magnetically assisted seeding of cells in the scaffold resulted most efficient for the 1% SPION concentration, providing good cell entrapment and adhesion rates. Mesenchymal Stromal Cells (MSCs) seeded onto PCL-HAp-1% SPION showed a good cell proliferation and intrinsic osteogenic potential, indicating no toxic effects of the employed scaffold materials. The performed characterizations and the collected set of data point on the inherent osteogenic potential of the newly developed PCL-HAp-1% SPION scaffolds, endorsing them towards next steps of in vitro and in vivo studies and validations.
AUTHOR Lotz, Benedict and Bothe, Friederike and Deubel, Anne-Kathrin and Hesse, Eliane and Renz, Yvonne and Werner, Carsten and Schäfer, Simone and Böck, Thomas and Groll, Jürgen and von Rechenberg, Brigitte and Richter, Wiltrud and Hagmann, Sebastien
Title Preclinical Testing of New Hydrogel Materials for Cartilage Repair: Overcoming Fixation Issues in a Large Animal Model [Abstract]
Year 2021
Journal/Proceedings International Journal of Biomaterials
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Abstract
Reinforced hydrogels represent a promising strategy for tissue engineering of articular cartilage. They can recreate mechanical and biological characteristics of native articular cartilage and promote cartilage regeneration in combination with mesenchymal stromal cells. One of the limitations of in vivo models for testing the outcome of tissue engineering approaches is implant fixation. The high mechanical stress within the knee joint, as well as the concave and convex cartilage surfaces, makes fixation of reinforced hydrogel challenging. Methods. Different fixation methods for full-thickness chondral defects in minipigs such as fibrin glue, BioGlue®, covering, and direct suturing of nonenforced and enforced constructs were compared. Because of insufficient fixation in chondral defects, superficial osteochondral defects in the femoral trochlea, as well as the femoral condyle, were examined using press-fit fixation. Two different hydrogels (starPEG and PAGE) were compared by 3D-micro-CT (μCT) analysis as well as histological analysis. Results. Our results showed fixation of below 50% for all methods in chondral defects. A superficial osteochondral defect of 1 mm depth was necessary for long-term fixation of a polycaprolactone (PCL)-reinforced hydrogel construct. Press-fit fixation seems to be adapted for a reliable fixation of 95% without confounding effects of glue or suture material. Despite the good integration of our constructs, especially in the starPEG group, visible bone lysis was detected in micro-CT analysis. There was no significant difference between the two hydrogels (starPEG and PAGE) and empty control defects regarding regeneration tissue and cell integration. However, in the starPEG group, more cell-containing hydrogel fragments were found within the defect area. Conclusion. Press-fit fixation in a superficial osteochondral defect in the medial trochlear groove of adult minipigs is a promising fixation method for reinforced hydrogels. To avoid bone lysis, future approaches should focus on multilayered constructs recreating the zonal cartilage as well as the calcified cartilage and the subchondral bone plate.
AUTHOR Chawla, Shikha and Desando, Giovanna and Gabusi, Elena and Sharma, Aarushi and Trucco, Diego and Chakraborty, Juhi and Manferdini, Cristina and Petretta, Mauro and Lisignoli, Gina and Ghosh, Sourabh
Title The effect of silk-gelatin bioink and TGF-β3 on mesenchymal stromal cells in 3D bioprinted chondrogenic constructs: A proteomic study [Abstract]
Year 2021
Journal/Proceedings Journal of Materials Research
Reftype Chawla2021
DOI/URL DOI
Abstract
Major limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin-gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.
AUTHOR Critchley, Susan and Sheehy, Eamon J. and Cunniffe, Gráinne and Diaz-Payno, Pedro and Carroll, Simon F. and Jeon, Oju and Alsberg, Eben and Brama, Pieter A. J. and Kelly, Daniel J.
Title 3D printing of fibre-reinforced cartilaginous templates for the regeneration of osteochondral defects [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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Abstract
Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage that is resistant to vascularization and endochondral ossification. During skeletal development articular cartilage also functions as a surface growth plate, which postnatally is replaced by a more spatially complex bone-cartilage interface. Motivated by this developmental process, the hypothesis of this study is that bi-phasic, fibre-reinforced cartilaginous templates can regenerate both the articular cartilage and subchondral bone within osteochondral defects created in caprine joints. To engineer mechanically competent implants, we first compared a range of 3D printed fibre networks (PCL, PLA and PLGA) for their capacity to mechanically reinforce alginate hydrogels whilst simultaneously supporting mesenchymal stem cell (MSC) chondrogenesis in vitro. These mechanically reinforced, MSC-laden alginate hydrogels were then used to engineer the endochondral bone forming phase of bi-phasic osteochondral constructs, with the overlying chondral phase consisting of cartilage tissue engineered using a co-culture of infrapatellar fat pad derived stem/stromal cells (FPSCs) and chondrocytes. Following chondrogenic priming and subcutaneous implantation in nude mice, these bi-phasic cartilaginous constructs were found to support the development of vascularised endochondral bone overlaid by phenotypically stable cartilage. These fibre-reinforced, bi-phasic cartilaginous templates were then evaluated in clinically relevant, large animal (caprine) model of osteochondral defect repair. Although the quality of repair was variable from animal-to-animal, in general more hyaline-like cartilage repair was observed after 6 months in animals treated with bi-phasic constructs compared to animals treated with commercial control scaffolds. This variability in the quality of repair points to the need for further improvements in the design of 3D bioprinted implants for joint regeneration. Statement of Significance Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage. In this study, we hypothesised that bi-phasic, fibre-reinforced cartilaginous templates could be leveraged to regenerate both the articular cartilage and subchondral bone within osteochondral defects. To this end we used 3D printed fibre networks to mechanically reinforce engineered transient cartilage, which also contained an overlying layer of phenotypically stable cartilage engineered using a co-culture of chondrocytes and stem cells. When chondrogenically primed and implanted into caprine osteochondral defects, these fibre-reinforced bi-phasic cartilaginous grafts were shown to spatially direct tissue development during joint repair. Such developmentally inspired tissue engineering strategies, enabled by advances in biofabrication and 3D printing, could form the basis of new classes of regenerative implants in orthopaedic medicine.
AUTHOR Mancini, I. A. D. and Schmidt, S. and Brommer, H. and Pouran, B. and Schäfer, S. and Tessmar, J. and Mensinga, A. and van Rijen, M. H. P. and Groll, J. and Blunk, T. and Levato, R. and Malda, J. and van Weeren, P. R.
Title A composite hydrogel-3D printed thermoplast osteochondral anchor as example for a zonal approach to cartilage repair: in vivo performance in a long-term equine model [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into the chondral part of the construct over which two layers of a thiol-ene cross-linkable hyaluronic acid/poly(glycidol) hybrid hydrogel (HA-SH/P(AGE-co-G)) were fabricated. The top layer contained Articular Cartilage Progenitor Cells (ACPCs) derived from the superficial layer of native cartilage tissue, the bottom layer contained mesenchymal stromal cells (MSCs). The chondral part of control constructs were homogeneously filled with MSCs. After six months in vivo, microtomography revealed significant bone growth into the anchor. Histologically, there was only limited production of cartilage-like tissue (despite persistency of hydrogel) both in zonal and non-zonal constructs. There were no differences in histological scoring; however, the repair tissue was significantly stiffer in defects repaired with zonal constructs. The sub-optimal quality of the repair tissue may be related to several factors, including early loss of implanted cells, or inappropriate degradation rate of the hydrogel. Nonetheless, this approach may be promising and research into further tailoring of biomaterials and of construct characteristics seems warranted.
AUTHOR Wang, Zehao and Hui, Aiping and Zhao, Hongbin and Ye, Xiaohan and Zhang, Chao and Wang, Aiqin and Zhang, Changqing
Title A Novel 3D-bioprinted Porous Nano Attapulgite Scaffolds with Good Performance for Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings International Journal of Nanomedicine
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BACKGROUND: Natural clay nanomaterials are an emerging class of biomaterial with great potential for tissue engineering and regenerative medicine applications, most notably for osteogenesis. MATERIALS AND METHODS: Herein, for the first time, novel tissue engineering scaffolds were prepared by 3D bioprinter using nontoxic and bioactive natural attapulgite (ATP) nanorods as starting materials, with polyvinyl alcohol as binder, and then sintered to obtain final scaffolds. The microscopic morphology and structure of ATP particles and scaffolds were observed by transmission electron microscope and scanning electron microscope. In vitro biocompatibility and osteogenesis with osteogenic precursor cell (hBMSCs) were assayed using MTT method, Live/Dead cell staining, alizarin red staining and RT-PCR. In vivo bone regeneration was evaluated with micro-CT and histology analysis in rat cranium defect model. RESULTS: We successfully printed a novel porous nano-ATP scaffold designed with inner channels with a dimension of 500 µm and wall structures with a thickness of 330 µm. The porosity of current 3D-printed scaffolds ranges from 75% to 82% and the longitudinal compressive strength was up to 4.32±0.52 MPa. We found firstly that nano-ATP scaffolds with excellent biocompatibility for hBMSCscould upregulate the expression of osteogenesis-related genes bmp2 and runx2 and calcium deposits in vitro. Interestingly, micro-CT and histology analysis revealed abundant newly formed bone was observed along the defect margin, even above and within the 3D bioprinted porous ATP scaffolds in a rat cranial defect model. Furthermore, histology analysis demonstrated that bone was formed directly following a process similar to membranous ossification without any intermediate cartilage formation and that many newly formed blood vessels are within the pores of 3D-printed scaffolds at four and eight weeks. CONCLUSION: These results suggest that the 3D-printed porous nano-ATP scaffolds are promising candidates for bone tissue engineering by osteogenesis and angiogenesis.
AUTHOR Diloksumpan, Paweena and de Ruijter, Myl{`{e}}ne and Castilho, Miguel and Gbureck, Uwe and Vermonden, Tina and van Weeren, P. Ren{'{e}} and Malda, Jos and Levato, Riccardo
Title Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.
AUTHOR Müller, Michael and Fisch, Philipp and Molnar, Marc and Eggert, Sebastian and Binelli, Marco and Maniura-Weber, Katharina and Zenobi-Wong, Marcy
Title Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s−1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.
AUTHOR Zhang, Hua and Cong, Yang and Osi, Amarachi Rosemary and Zhou, Yang and Huang, Fangcheng and Zaccaria, Remo P. and Chen, Jing and Wang, Rong and Fu, Jun
Title Direct 3D Printed Biomimetic Scaffolds Based on Hydrogel Microparticles for Cell Spheroid Growth [Abstract]
Year 2020
Journal/Proceedings Advanced Functional Materials
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Abstract Biocompatible hydrogel inks with shear-thinning, appropriate yield strength, and fast self-healing are desired for 3D bioprinting. However, the lack of ideal 3D bioprinting inks with outstanding printability and high structural fidelity, as well as cell-compatibility, has hindered the progress of extrusion-based 3D bioprinting for tissue engineering. In this study, novel self-healable pre-cross-linked hydrogel microparticles (pcHμPs) of chitosan methacrylate (CHMA) and polyvinyl alcohol (PVA) hybrid hydrogels are developed and used as bioinks for extrusion-based 3D printing of scaffolds with high fidelity and biocompatibility. The pcHμPs display excellent shear thinning when injected through a syringe and subsequently self-heal into gels as shear forces are removed. Numerical simulations indicate that the pcHμPs experience a plug flow in the nozzle with minimal disturbance, which favors a steady and continuous printing. Moreover, the pcHμPs show a self-supportive yield strength (540 Pa), which is critical for the fidelity of printed constructs. A series of biomimetic constructs with very high aspect ratio and delicate fine structures are directly printed by using the pcHμP ink. The 3D printed scaffolds support the growth of bone-marrow-derived mesenchymal stem cells and formation of cell spheroids, which are most important for tissue engineering.
AUTHOR Abu Awwad, Hosam Al-Deen M. and Thiagarajan, Lalitha and Kanczler, Janos M. and Amer, Mahetab H. and Bruce, Gordon and Lanham, Stuart and Rumney, Robin M. H. and Oreffo, Richard O. C. and Dixon, James E.
Title Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair [Abstract]
Year 2020
Journal/Proceedings Journal of Controlled Release
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Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.
AUTHOR Eltaher, Hoda M. and Abukunna, Fatima E. and Ruiz-Cantu, Laura and Stone, Zack and Yang, Jing and Dixon, James E.
Title Human-scale tissues with patterned vascular networks by additive manufacturing of sacrificial sugar-protein composites [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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Abstract
Combating necrosis, by supplying nutrients and removing waste, presents the major challenge for engineering large three-dimensional (3D) tissues. Previous elegant work used 3D printing with carbohydrate glass as a cytocompatible sacrificial template to create complex engineered tissues with vascular networks (Miller et al. 2012, Nature Materials). The fragile nature of this material compounded with the technical complexity needed to create high-resolution structures led us to create a flexible sugar-protein composite, termed Gelatin-sucrose matrix (GSM), to achieve a more robust and applicable material. Here we developed a low-range (25–37˚C) temperature sensitive formulation that can be moulded with micron-resolution features or cast during 3D printing to produce complex flexible filament networks forming sacrificial vessels. Using the temperature-sensitivity, we could control filament degeneration meaning GSM can be used with a variety of matrices and crosslinking strategies. Furthermore by incorporation of biocompatible crosslinkers into GSM directly, we could create thin endothelialized vessel walls and generate patterned tissues containing multiple matrices and cell-types. We also demonstrated that perfused vascular channels sustain metabolic function of a variety of cell-types including primary human cells. Importantly, we were able to construct vascularized human noses which otherwise would have been necrotic. Our material can now be exploited to create human-scale tissues for regenerative medicine applications. Statement of Significance Authentic and engineered tissues have demands for mass transport, exchanging nutrients and oxygen, and therefore require vascularization to retain viability and inhibit necrosis. Basic vascular networks must be included within engineered tissues intrinsically. Yet, this has been unachievable in physiologically-sized constructs with tissue-like cell densities until recently. Sacrificial moulding is an alternative in which networks of rigid lattices of filaments are created to prevent subsequent matrix ingress. Our study describes a biocompatible sacrificial sugar-protein formulation; GSM, made from mixtures of inexpensive and readily available bio-grade materials. GSM can be cast/moulded or bioprinted as sacrificial filaments that can rapidly dissolve in an aqueous environment temperature-sensitively. GSM material can be used to engineer viable and vascularized human-scale tissues for regenerative medicine applications.
AUTHOR Hauptstein, Julia and Böck, Thomas and Bartolf-Kopp, Michael and Forster, Leonard and Stahlhut, Philipp and Nadernezhad, Ali and Blahetek, Gina and Zernecke-Madsen, Alma and Detsch, Rainer and Jüngst, Tomasz and Groll, Jürgen and Teßmar, Jörg and Blunk, Torsten
Title Hyaluronic Acid-Based Bioink Composition Enabling 3D Bioprinting and Improving Quality of Deposited Cartilaginous Extracellular Matrix [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.
AUTHOR De Moor, Lise and Fernandez, Sélina and Vercruysse, Chris and Tytgat, Liesbeth and Asadian, Mahtab and De Geyter, Nathalie and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids [Abstract]
Year 2020
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
AUTHOR Figueiredo, Lara and Le Visage, Catherine and Weiss, Pierre and Yang, Jing
Title Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks [Abstract]
Year 2020
Journal/Proceedings Polymers
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Abstract
The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.
AUTHOR Schipani, Rossana and Scheurer, Stefan and Florentin, Romain and Critchley, Susan E. and Kelly, Daniel John
Title Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.
AUTHOR Sanz-Fraile, Hector and Amorós, Susana and Mendizabal, Irene Isabel and Gálvez-Montón, Carolina and Prat-Vidal, Cristina and Bayés-Genís, Antoni and Navajas, Daniel and Farre, Ramon and Otero, Jorge
Title Silk-reinforced Collagen Hydrogels with Raised Multiscale Stiffness for Mesenchymal Cells 3D Culture [Abstract]
Year 2020
Journal/Proceedings Tissue Engineering Part A
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Abstract
Type I collagen hydrogels are of high interest in tissue engineering. With the evolution of 3D bioprinting technologies, a high number of collagen-based scaffolds have been reported for the development of 3D cell cultures. A recent proposal was to mix collagen with silk fibroin derived from Bombyx Mori silkworm. Nevertheless, due to the difficulties in the preparation and the characteristics of the protein, several problems like phase separation and collagen denaturation appears during the procedure. Therefore, the common solution is to diminish the concentration of collagen although in that way the most biologically relevant component is reduced. In the present work, we present a new, simple and effective method to develop a collagen-silk hybrid hydrogel with high collagen concentration and with increased stiffness approaching that of natural tissues, which could be of high interest for the development of cardiac patches for myocardial regeneration and for preconditioning of mesenchymal stem cells to improve their therapeutic potential. Sericin in the silk was preserved by using a physical solubilizing procedure which results in a preserved fibrous structure of type I collagen, as shown by ultrastructural imaging. The macro- and micromechanical properties of the hybrid hydrogels measured by tensile stretch and Atomic Force Microscopy respectively, showed a more than two-fold stiffening as compared with collagen-only hydrogels. Rheological measurements showed improved printability properties for the developed biomaterial. The suitability of the hydrogels for 3D cell culture was assessed by 3D bioprinting bone marrow-derived mesenchymal stem cells cultured within the scaffolds. The result was a biomaterial with improved printability characteristics that better resembled the mechanical properties of natural soft tissues while preserving biocompatibility owing to the high concentration of collagen.
AUTHOR Schwab, Andrea and Helary, Christophe and Richards, Geoff and Alini, Mauro and Eglin, David and D{textquoteright}Este, Matteo
Title Tissue mimetic hyaluronan bio-ink containing oriented collagen fibers to modulate hMSC spreading and differentiation [Abstract]
Year 2020
Journal/Proceedings bioRxiv
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Abstract
Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes, chemical and biological gradients. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the sub-micron and the molecular level.In this study, we introduce a (bio)ink containing hyaluronan (HA) as tyramine derivative (THA) and collagen (Col). Similarly to other connective tissues, in this (bio)ink Col is present in fibrillar form and HA as viscoelastic space filler. THA was enzymatically crosslinked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. THA-Col composite displayed synergistic properties in terms of storage modulus and shear-thinning, translating into good printability.Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second harmonic generation.Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on cell behavior and chondrogenic differentiation.THA-Col showed cell instructive properties modulating hMSC adhesion, morphology and sprouting from spheroids stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded into aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and matrix production, hMSCs embedded into the bioink displayed chondrogenic differentiation comparable or superior to standard pellet culture by means of proteoglycan production (Safranin O staining and proteoglycan quantification) as well as increase in cartilage related genes.The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.
AUTHOR Nasim Golafshan and Elke Vorndran and Stefan Zaharievski and Harold Brommer and Firoz Babu Kadumudi and Alireza Dolatshahi-Pirouz and Uwe Gbureck and René {van Weeren} and Miguel Castilho and Jos Malda
Title Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model [Abstract]
Year 2020
Journal/Proceedings Biomaterials
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Abstract
One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr2+) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr2+ ions (MgPSr) and a medical grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm3) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load bearing properties of MgP-based ceramics with simultaneously induction of new bone formation.
AUTHOR Freeman, F. E. and Browe, D. C. and Nulty, J. and Von Euw, S. and Grayson, W. L. and Kelly, D. J.
Title Biofabrication of multiscale bone extracellular matrix scaffolds for bone tissue engineering. [Abstract]
Year 2019
Journal/Proceedings European Cells and Materials Journal
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Abstract
Interconnected porosity is critical to the design of regenerative scaffolds, as it permits cell migration, vascularisation and diffusion of nutrients and regulatory molecules inside the scaffold. 3D printing is a promising strategy to achieve this as it allows the control over scaffold pore size, porosity and interconnectivity. Thus, the aim of the present study was to integrate distinct biofabrication strategies to develop a multiscale porous scaffold that was not only mechanically functional at the time of implantation, but also facilitated rapid vascularisation and provided stem cells with appropriate cues to enable their differentiation into osteoblasts. To achieve this, polycaprolactone (PCL) was functionalised with decellularised bone extracellular matrix (ECM), to produce osteoinductive filaments for 3D printing. The addition of bone ECM to the PCL not only increased the mechanical properties of the resulting scaffold, but also increased cellular attachment and enhanced osteogenesis of mesenchymal stem cells (MSCs). In vivo, scaffold pore size determined the level of vascularisation, with a larger filament spacing supporting faster vessel in-growth and more new bone formation. By freeze-drying solubilised bone ECM within these 3D-printed scaffolds, it was possible to introduce a matrix network with microscale porosity that further enhanced cellular attachment in vitro and increased vessel infiltration and overall levels of new bone formation in vivo. To conclude, an "off-the-shelf" multiscale bone-ECM-derived scaffold was developed that was mechanically stable and, once implanted in vivo, will drive vascularisation and, ultimately, lead to bone regeneration.
AUTHOR Cofiño, Carla and Perez-Amodio, Soledad and Semino, Carlos E. and Engel, Elisabeth and Mateos-Timoneda, Miguel A.
Title Development of a Self-Assembled Peptide/Methylcellulose-Based Bioink for 3D Bioprinting [Abstract]
Year 2019
Journal/Proceedings Macromolecular Materials and Engineering
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Abstract
Abstract The introduction of 3D bioprinting to fabricate living constructs with tailored architecture has provided a new paradigm for biofabrication, with the potential to overcome several drawbacks of conventional scaffold-based tissue regeneration strategies. Hydrogel-based materials are suitable candidates regarding cell biocompatibility but often display poor mechanical properties. Self-assembling peptides are a promising source of biomaterials to be used as 3D scaffolds based on their similarity to extracellular matrices (structurally and mechanically). In this study, an advanced bioink for biofabrication is presented based on the optimization of a RAD16-I-based biomaterial. The strategy followed to build 3D predefined structures by 3D printing is based on an enhancement of bioink viscosity by adding methylcellulose (MC) to a RAD16-I solution. The resultant constructs display high shape fidelity and stability and embedded human mesenchymal stem cells present high viability after 7 days of culture. Moreover, cells are also able to differentiate to the adipogenic lineage, suggesting the suitability of this novel biomaterial for soft tissue engineering applications.
AUTHOR Rathan, Swetha and Dejob, Léa and Schipani, Rossana and Haffner, Benjamin and Möbius, Matthias E. and Kelly, Daniel J.
Title Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering [Abstract]
Year 2019
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct “print-and-implant” cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.
AUTHOR Sharma, Aarushi and Desando, Giovanna and Petretta, Mauro and Chawla, Shikha and Bartolotti, Isabella and Manferdini, Cristina and Paolella, Francesca and Gabusi, Elena and Trucco, Diego and Ghosh, Sourabh and Lisignoli, Gina
Title Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells
Year 2019
Journal/Proceedings ACS Biomaterials Science & Engineering
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AUTHOR Xu, Yichi and Peng, Jiang and Richards, Geoff and Lu, Shibi and Eglin, David
Title Optimization of electrospray fabrication of stem cell–embedded alginate–gelatin microspheres and their assembly in 3D-printed poly(ε-caprolactone) scaffold for cartilage tissue engineering [Abstract]
Year 2019
Journal/Proceedings Journal of Orthopaedic Translation
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Abstract
Objective Our study reports the optimization of electrospray human bone marrow stromal cell (hBMSCs)–embedded alginate–gelatin (Alg-Gel, same as following) microspheres for the purpose of their assembly in 3D-printed poly(ε-caprolactone) (PCL) scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct. Methods The fabrication of the Alg-Gel microspheres using an electrospray technique was optimized in terms of polydispersity, yield of microspheres and circularity and varying fabrication conditions. PCL scaffolds were designed and printed by melt extrusion. Then, four groups were set: Alg-hBMSC microspheres cultured in the 2D well plate (Alg-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres embedded in PCL scaffold cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group and Alg-Gel-hBMSCs microspheres cultured in the 3D bioreactor (Alg-Gel-hBMSCs+3D) group. Cell viability, proliferation and chondrogenic differentiation were evaluated, and mechanical test was performed. Results Nonaggregated, low polydispersity and almost spherical microspheres of average diameter of 200–300 μm were produced with alginate 1.5 w: v%, gelatin (Type B) concentration of 0.5 w: v % and CaCl2 coagulating bath concentration of 3.0 w: v %, using 30G needle size and 8 kV and 0.6 bar voltage and air pressure, respectively. Alginate with gelatin hydrogel improved viability and promoted hBMSC proliferation better than alginate microspheres. Interestingly, hBMSCs embedded in microspheres assembled in 3D-printed PCL scaffold and cultured in a 3D bioreactor were more proliferative in comparison to the previous two groups (p < 0.05). Similarly, the GAG content, GAG/DNA ratio as well as Coll 2 and Aggr gene expression were increased in the last two groups. Conclusion Optimization of hBMSC-embedded Alg-Gel microspheres produced by electrospray has been performed. The Alg-Gel composition selected allows conservation of hBMSC viability and supports proliferation and matrix deposition. The possibility to seed and assemble microspheres in designed 3D-printed PCL scaffolds for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct was demonstrated. Translational potential of this article We optimize and demonstrate that electrospray microsphere fabrication is a cytocompatible and facile process to produce the hBMSC-embedded microsize tissue-like particles that can easily be assembled into a stable construct. This finding could have application in the development of mechanically competent stem cell–based tissue engineering of cartilage regeneration.
AUTHOR Filardo, G. and Petretta, M. and Cavallo, C. and Roseti, L. and Durante, S. and Albisinni, U. and Grigolo, B.
Title Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold [Abstract]
Year 2019
Journal/Proceedings Bone and Joint Research
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Abstract
Objectives Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.
AUTHOR Gloria, Antonio and Frydman, B. and Lamas, Miguel L. and Serra, Armenio C. and Martorelli, Massimo and Coelho, Jorge F. J. and Fonseca, Ana C. and Domingos, M.
Title The influence of poly(ester amide) on the structural and functional features of 3D additive manufactured poly(ε-caprolactone) scaffolds [Abstract]
Year 2019
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
The current research reports for the first time the use of blends of poly(ε-caprolactone) (PCL) and poly(ester amide) (PEA) for the fabrication of 3D additive manufactured scaffolds. Tailor made PEA was synthesized to afford fully miscible blends of PCL and PEA using different percentages (5, 10, 15 and 20% w/w). Stability, characteristic temperatures and material's compatibility were studied through thermal analyses (i.e., TGA, DSC). Even though DMTA and static compression tests demonstrated the possibility to improve the storage modulus, Young's modulus and maximum stress by increasing the amount of PEA, a decrease of hardness was found beyond a threshold concentration of PEA as the lowest values were achieved for PCL/PEA (20% w/w) scaffolds (from 0.39 ± 0.03 GPa to 0.21 ± 0.02 GPa in the analysed load range). The scaffolds presented a controlled morphology and a fully interconnected network of internal channels. The water contact angle measurements showed a clear increase of hydrophilicity resulting from the addition of PEA. This result was further corroborated with the improved adhesion and proliferation of human mesenchymal stem cells (hMSCs). The presence of PEA also influenced the cell morphology. Better cell spreading and a much higher and homogenous number of cells were observed for PCL/PEA scaffolds when compared to PCL ones.
AUTHOR Petta, D. and Armiento, A. R. and Grijpma, D. and Alini, M. and Eglin, D. and D'Este, M.
Title 3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.
AUTHOR Tognato, Riccardo and Armiento, Angela R. and Bonfrate, Valentina and Levato, Riccardo and Malda, Jos and Alini, Mauro and Eglin, David and Giancane, Gabriele and Serra, Tiziano
Title A Stimuli-Responsive Nanocomposite for 3D Anisotropic Cell-Guidance and Magnetic Soft Robotics [Abstract]
Year 2018
Journal/Proceedings Advanced Functional Materials
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Abstract
Abstract Stimuli-responsive materials have the potential to enable the generation of new bioinspired devices with unique physicochemical properties and cell-instructive ability. Enhancing biocompatibility while simplifying the production methodologies, as well as enabling the creation of complex constructs, i.e., via 3D (bio)printing technologies, remains key challenge in the field. Here, a novel method is presented to biofabricate cellularized anisotropic hybrid hydrogel through a mild and biocompatible process driven by multiple external stimuli: magnetic field, temperature, and light. A low-intensity magnetic field is used to align mosaic iron oxide nanoparticles (IOPs) into filaments with tunable size within a gelatin methacryloyl matrix. Cells seeded on top or embedded within the hydrogel align to the same axes of the IOPs filaments. Furthermore, in 3D, C2C12 skeletal myoblasts differentiate toward myotubes even in the absence of differentiation media. 3D printing of the nanocomposite hydrogel is achieved and creation of complex heterogeneous structures that respond to magnetic field is demonstrated. By combining the advanced, stimuli-responsive hydrogel with the architectural control provided by bioprinting technologies, 3D constructs can also be created that, although inspired by nature, express functionalities beyond those of native tissue, which have important application in soft robotics, bioactuators, and bionic devices.
AUTHOR Mouser, Vivian H. M. and Levato, Riccardo and Mensinga, Anneloes and Dhert, Wouter J. A. and Gawlitta, Debby and Malda, Jos
Title Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs [Abstract]
Year 2018
Journal/Proceedings Connective Tissue Research
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Abstract
ABSTRACTBioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks.The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation.ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation.
AUTHOR Prasopthum, Aruna and Shakesheff, Kevin M. and Yang, Jing
Title Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Abstract
Three-dimensional (3D) printing is a powerful manufacturing tool for making 3D structures with well-defined architectures for a wide range of applications. The field of tissue engineering has also adopted this technology to fabricate scaffolds for tissue regeneration. The ability to control architecture of scaffolds, e.g. matching anatomical shapes and having defined pore size, has since been improved significantly. However, the material surface of these scaffolds is smooth and does not resemble that found in natural extracellular matrix (ECM), in particular, the nanofibrous morphology of collagen. This natural nanoscale morphology plays a critical role in cell behaviour. Here, we have developed a new approach to directly fabricate polymeric scaffolds with an ECM-like nanofibrous topography and defined architectures using extrusion-based 3D printing. 3D printed tall scaffolds with interconnected pores were created with disparate features spanning from nanometres to centimetres. Our approach removes the need for a sacrificial mould and subsequent mould removal compared to previous methods. Moreover, the nanofibrous topography of the 3D printed scaffolds significantly enhanced protein absorption, cell adhesion and differentiation of human mesenchymal stem cells when compared to those with smooth material surfaces. These 3D printed scaffolds with both defined architectures and nanoscale ECM-mimicking morphologies have potential applications in cartilage and bone regeneration.
AUTHOR Stichler, Simone and Böck, Thomas and Paxton, Naomi Claire and Bertlein, Sarah and Levato, Riccardo and Schill, Verena and Smolan, Willi and Malda, Jos and Tessmar, Joerg and Blunk, Torsten and Groll, Juergen
Title Double printing of hyaluronic acid / poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Abstract This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. Chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by UV-induced radical thiol-ene coupling between the thiol and allyl groups. Addition of unmodified high molecular weight HA (1.36 MDa) allowed tuning of the rheology for extrusion based bioprinting. Incorporation of additional HA resulted in hydrogels with lower Young’s modulus and higher swelling ratio especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanical stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with the thermoplastic poly--caprolactone (PCL).
AUTHOR Henriksson, I. and Gatenholm, P. and Hägg, D. A.
Title Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds [Abstract]
Year 2017
Journal/Proceedings Biofabrication
Reftype
DOI/URL URL
Abstract
Compared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPAR γ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.
AUTHOR Paxton, Naomi Claire and Smolan, Willi and Böck, Thomas and Melchels, Ferry P. W. and Groll, Juergen and Juengst, Tomasz
Title Proposal to Assess Printability of Bioinks for Extrusion-Based Bioprinting and Evaluation of Rheological Properties Governing Bioprintability [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Abstract The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application, but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially-available crème, poloxamer 407, alginate-based inks and an alginate-gelatin composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
AUTHOR Levato, Riccardo and Webb, William R. and Otto, Iris A. and Mensinga, Anneloes and Zhang, Yadan and van Rijen, Mattie and van Weeren, René and Khan, Ilyas M. and Malda, Jos
Title The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells
Year 2017
Journal/Proceedings Acta Biomaterialia
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AUTHOR Freeman, Fiona E. and Kelly, Daniel J.
Title Tuning Alginate Bioink Stiffness and Composition for Controlled Growth Factor Delivery and to Spatially Direct MSC Fate within Bioprinted Tissues [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
Reftype Freeman2017
DOI/URL DOI
Abstract
Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young’s Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.
AUTHOR Daly, Andrew C. and Cunniffe, Gr{'{a}}inne M. and Sathy, Binulal N. and Jeon, Oju and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering [Abstract]
Year 2016
Journal/Proceedings Advanced Healthcare Materials
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Abstract
The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo.
AUTHOR Daly, Andrew C. and Critchley, Susan E. and Rencsok, Emily M. and Kelly, Daniel J.
Title A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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DOI/URL