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AUTHOR Terpstra, Margo L. and Li, Jinyu and Mensinga, Anneloes and de Ruijter, Myl{`{e}}ne and van Rijen, Mattie H. P. and Androulidakis, Charalampos and Galiotis, Costas and Papantoniou, Ioannis and Matsusaki, Michiya and Malda, Jos and Levato, Riccardo
Title Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material three-dimensional (3D) bioprinting strategies have the potential to resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge to date. In this study, we developed endothelial cell-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM), respectively. Human umbilical vein endothelial cell (HUVEC)-driven capillary networks started to form 2 d after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29%, and the number of microvessel junctions by 37% after 14 d, compared to bioinks with pro-angiogenic col-1 MFs. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a gellan gum microgel suspension bath. These 3D meniscus-like constructs were cultured up to 14 d, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models of vascular-to-avascular tissue interfaces, cancer progression, and for testing anti-angiogenic therapies.
AUTHOR Dufour, A. and Gallostra, X. Barceló and O'Keeffe, C. and Eichholz, K. and Von Euw, S. and Garcia, O. and Kelly, D. J.
Title Integrating melt electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage [Abstract]
Year 2022
Journal/Proceedings Biomaterials
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Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjet printing was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50 mm × 50 mm) cartilage implants containing over 3,500 cellular aggregates in under 15 min. After 8 weeks in culture, a 50-fold increase in the compressive stiffness of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.
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 Liu, Chun and Dai, Ting and Wu, Xiaoyu and Ma, Jiayi and Liu, Jun and Wu, Siyu and Yang, Lei and Zhao, Hongbin
Title 3D bioprinting of cell-laden nano-attapulgite/gelatin methacrylate composite hydrogel scaffolds for bone tissue repair [Abstract]
Year 2023
Journal/Proceedings Journal of Materials Science & Technology
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Bone tissue engineering (BTE) has proven to be a promising strategy for bone defect repair. Due to its excellent biological properties, gelatin methacrylate (GelMA) hydrogels have been used as bioinks for 3D bioprinting in some BTE studies to produce scaffolds for bone regeneration. However, applications for load-bearing defects are limited by poor mechanical properties and a lack of bioactivity. In this study, 3D printing technology was used to create nano-attapulgite (nano-ATP)/GelMA composite hydrogels loaded into mouse bone mesenchymal stem cells (BMSCs) and mouse umbilical vein endothelial cells (MUVECs). The bioprintability, physicochemical properties, and mechanical properties were all thoroughly evaluated. Our findings showed that nano-ATP groups outperform the control group in terms of printability, indicating that nano-ATP is beneficial for printability. Additionally, after incorporation with nano-ATP, the mechanical strength of the composite hydrogels was significantly improved, resulting in adequate mechanical properties for bone regeneration. The presence of nano-ATP in the scaffolds has also been studied for cell-material interactions. The findings show that cells within the scaffold not only have high viability but also a clear proclivity to promote osteogenic differentiation of BMSCs. Besides, the MUVECs-loaded composite hydrogels demonstrated increased angiogenic activity. A cranial defect model was also developed to evaluate the bone repair capability of scaffolds loaded with rat BMSCs. According to histological analysis, cell-laden nano-ATP composite hydrogels can effectively improve bone regeneration and promote angiogenesis. This study demonstrated the potential of nano-ATP for bone tissue engineering, which should also increase the clinical practicality of nano-ATP.
AUTHOR Pitacco, Pierluca and Sadowska, Joanna M. and O'Brien, Fergal J. and Kelly, Daniel J.
Title 3D bioprinting of cartilaginous templates for large bone defect healing [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. Statement of significance Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.
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 Man, Kenny and Barroso, Inês A. and Brunet, Mathieu Y. and Peacock, Ben and Federici, Angelica S. and Hoey, David A. and Cox, Sophie C.
Title Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair [Abstract]
Year 2022
Journal/Proceedings International Journal of Molecular Sciences
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Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs’ potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.
AUTHOR Rahimnejad, Maedeh and Adoungotchodo, Atma and Demarquette, Nicole R. and Lerouge, Sophie
Title FRESH bioprinting of biodegradable chitosan thermosensitive hydrogels [Abstract]
Year 2022
Journal/Proceedings Bioprinting
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Thermosensitive chitosan (CH)-based hydrogels prepared with a mix of sodium bicarbonate and β-glycerophosphate as gelling agents rapidly pass from a liquid at room temperature to a mechanically strong solid at body temperature without any crosslinker. They show excellent potential for tissue engineering applications and could be interesting candidates for bioprinting. Unfortunately, since gelation is not instantaneous, formulations compatible with cell encapsulation (chitosan concentrations around 2% or lower) lead to very poor resolution and fidelity due to filament spreading. Here, we investigate the FRESH bioprinting approach with a warm sacrificial support bath, to overcome these limitations and enhance their bioprintability. First, a support bath, made of Pluronic including sodium chloride salt as a rheology modifier agent, was designed to meet the specific physical state requirements (solid at 37 °C and liquid at room temperature) and rheological properties appropriate for bioprinting. This support bath presented yield stress of over 100 Pa, a shear thinning behavior, and fast self-healing during cyclic recovery tests. Three different chitosan hydrogels (CH2%w/v, CH3%w/v, and a mixture of CH and gelatin) were tested for their ability to form filament and 3D structures, with and without a support bath. Both the resolution and mechanical properties of the printed structure were drastically enhanced using the FRESH method, with an approximate four fold decrease of the filament diameter which is close to the needle diameter. The printed structures were easily harvested without altering their shape by cooling down the support bath, and do not swell when immersed in PBS. Live/dead assays confirmed that the viability of encapsulated mesenchymal stem cells was highest in CH2% and that the support bath-assisted bioprinting process did not adversely impact cell viability. This study demonstrates that using a warm FRESH-like approach drastically enhances the potential for bioprinting of the thermosensitive biodegradable chitosan hydrogels and opens up a wide range of applications for 3D models and tissue engineering.
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 Staubli, Flurina and Stoddart, Martin J. and D'Este, Matteo and Schwab, Andrea
Title Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. Statement of significance In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.
AUTHOR Eichholz, Kian and Freeman, Fiona and Pitacco, Pierluca and Nulty, Jessica and Ahern, Daniel and Burdis, Ross and Browe, David and Garcia, Orquidea and Hoey, David and Kelly, Daniel John
Title Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Abstract
Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineered constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeks in vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone tissue engineering strategies.
AUTHOR Burdis, Ross and Chariyev-Prinz, Farhad and Browe, David C. and Freeman, Fiona E. and Nulty, Jessica and McDonnell, Emily E. and Eichholz, Kian F. and Wang, Bin and Brama, Pieter and Kelly, Daniel J.
Title Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing [Abstract]
Year 2022
Journal/Proceedings Biomaterials
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Abstract
Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-β accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.
AUTHOR Hatt, Luan P. and Armiento, Angela R. and Mys, Karen and Thompson, Keith and Hildebrand, Maria and Nehrbass, Dirk and Müller, Werner E. G. and Zeiter, Stephan and Eglin, David and Stoddart, Martin J.
Title Standard in vitro evaluations of engineered bone substitutes are not sufficient to predict in vivo preclinical model outcomes [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Abstract
Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non–unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow – derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. Statement of significance Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.
AUTHOR Anderson, Margaret and Dubey, Nileshkumar and Bogie, Kath and Cao, Chen and Li, Junying and Lerchbacker, Joseph and Mendonça, Gustavo and Kauffmann, Frederic and Bottino, Marco C. and Kaigler, Darnell
Title Three-dimensional printing of clinical scale and personalized calcium phosphate scaffolds for alveolar bone reconstruction [Abstract]
Year 2022
Journal/Proceedings Dental Materials
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Abstract
Objective Alveolar bone defects can be highly variable in their morphology and, as the defect size increases, they become more challenging to treat with currently available therapeutics and biomaterials. This investigation sought to devise a protocol for fabricating customized clinical scale and patient-specific, bioceramic scaffolds for reconstruction of large alveolar bone defects. Methods Two types of calcium phosphate (CaP)-based bioceramic scaffolds (alginate/β-TCP and hydroxyapatite/α-TCP, hereafter referred to as hybrid CaP and Osteoink™, respectively) were designed, 3D printed, and their biocompatibility with alveolar bone marrow stem cells and mechanical properties were determined. Following scaffold optimization, a workflow was developed to use cone beam computed tomographic (CBCT) imaging to design and 3D print, defect-specific bioceramic scaffolds for clinical-scale bone defects. Results Osteoink™ scaffolds had the highest compressive strength when compared to hybrid CaP with different infill orientation. In cell culture medium, hybrid CaP degradation resulted in decreased pH (6.3) and toxicity to stem cells; however, OsteoInk™ scaffolds maintained a stable pH (7.2) in culture and passed the ISO standard for cytotoxicity. Finally, a clinically feasible laboratory workflow was developed and evaluated using CBCT imaging to engineer customized and defect-specific CaP scaffolds using OsteoInk™. It was determined that printed scaffolds had a high degree of accuracy to fit the respective clinical defects for which they were designed (0.27 mm morphological deviation of printed scaffolds from digital design). Significance From patient to patient, large alveolar bone defects are difficult to treat due to high variability in their complex morphologies and architecture. Our findings shows that Osteoink™ is a biocompatible material for 3D printing of clinically acceptable, patient-specific scaffolds with precision-fit for use in alveolar bone reconstructive procedures. Collectively, emerging digital technologies including CBCT imaging, 3D surgical planning, and (bio)printing can be integrated to address this unmet clinical challenge.
AUTHOR Barceló, Xavier and Eichholz, Kian F. and Garcia, Orquidea and Kelly, Daniel J.
Title Tuning the Degradation Rate of Alginate-Based Bioinks for Bioprinting Functional Cartilage Tissue [Abstract]
Year 2022
Journal/Proceedings Biomedicines
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Abstract
Negative foreign body responses following the in vivo implantation of bioprinted implants motivate the development of novel bioinks which can rapidly degrade with the formation of functional tissue, whilst still maintaining desired shapes post-printing. Here, we investigated the oxidation of alginate as a means to modify the degradation rate of alginate-based bioinks for cartilage tissue engineering applications. Raw and partially oxidized alginate (OA) were combined at different ratios (Alginate:OA at 100:0; 75:25; 50:50; 25:75; 0:100) to provide finer control over the rate of bioink degradation. These alginate blends were then combined with a temporary viscosity modifier (gelatin) to produce a range of degradable bioinks with rheological properties suitable for extrusion bioprinting. The rate of degradation was found to be highly dependent on the OA content of the bioink. Despite this high mass loss, the initially printed geometry was maintained throughout a 4 week in vitro culture period for all bioink blends except the 0:100 group. All bioink blends also supported robust chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs), resulting in the development of a hyaline-like tissue that was rich in type II collagen and negative for calcific deposits. Such tuneable inks offer numerous benefits to the field of 3D bioprinting, from providing space in a controllable manner for new extracellular matrix deposition, to alleviating concerns associated with a foreign body response to printed material inks 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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Abstract
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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Abstract
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
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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Abstract
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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Abstract
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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Abstract
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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Abstract
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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Abstract
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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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 Paindelli, Claudia and Casarin, Stefano and Wang, Feng and Diaz-Gomez, Luis and Zhang, Jianhua and Mikos, Antonios G. and Logothetis, Christopher J. and Friedl, Peter and Dondossola, Eleonora
Title Enhancing Radium 223 treatment efficacy by anti-beta 1 integrin targeting [Abstract]
Year 2021
Journal/Proceedings Journal of Nuclear Medicine
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Abstract
Radium 223 (223Ra) is an α-emitter approved for the treatment of bone metastatic prostate cancer (PCa), which exerts direct cytotoxicity towards PCa cells near the bone interface, whereas cells positioned in the core respond poorly, due to short α-particle penetrance. β1 integrin (β1I) interference has been shown to increase radiosensitivity and significantly enhance external beam radiation efficiency. We hypothesized that targeting β1I would improve 223Ra outcome. We tested the effect of combining 223Ra and anti-β1I antibody treatment in PC3 and C4-2B PCa cell models expressing high and low β1I levels, respectively. In vivo tumor growth was evaluated through bioluminescence. Cellular and molecular determinants of response were analyzed by ex vivo three-dimensional imaging of bone lesions, proteomic analysis and further confirmed by computational modeling and in vitro functional analysis in tissue-engineered bone mimetic systems. Interference with β1I combined with 223Ra reduced PC3 cell growth in bone and significantly improved overall mouse survival, while no change was achieved in C4-2B tumors. Anti-β1I treatment decreased PC3 tumor cell mitosis index and spatially expanded 223Ra lethal effects two-fold, in vivo and in silico. Regression was paralleled by decreased expression of radio-resistance mediators. Targeting β1I significantly improves 223Ra outcome and points towards combinatorial application in PCa tumors with high β1I expression.
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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Abstract
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
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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Abstract
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 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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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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