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Synthetic Bone‐Like Structures Through Omnidirectional Ceramic Bioprinting in Cell Suspensions

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Abstract and Figures

The integration of hierarchical structure, chemistry, and functional activity within tissue‐engineered scaffolds is of great importance in mimicking native bone tissue. Bone is a highly mineralized tissue which forms at ambient conditions by continuous crystallization of the mineral phase within an organic matrix in the presence of bone residing cells. Despite recent advances in the biofabrication of complex engineered tissues, replication of the heterogeneity of bone microenvironments has been a major challenge in constructing biomimetic bone scaffolds. Herein, inspired by the bone biomineralization process, the first example of bone mimicking constructs by 3D writing of a novel apatite‐transforming ink in a supportive microgel matrix with living cells is demonstrated. Using this technique, complex bone‐mimicked constructs are made at room temperature without requiring invasive chemicals, radiation, or postprocessing steps. This study demonstrates that mineralized constructs can be deposited within a high density of stem cells, directing the cellular organization, and promoting osteogenesis in vitro. These findings offer a new strategy for fabrication of bone mimicking constructs for bone tissue regeneration with scope to generate custom bone microenvironments for disease modeling, multicellular delivery, and in vivo bone repair.
A) Free‐form writing the CaP‐ink in a suspension of gelatin microspheres with properties of a yield‐stress fluid; 3D printing of complex structures versus structures generated by stacking of 2D monolayers. B) Representative image of direct extrusion of CaP‐ink in culture media (Pore size: ≈500 and 1500 µm (X‐Y plane) and 250 µm (Z plane), and 100% interconnectivity between the pores) and scanning electron micrographs demonstrating the nanostructured interface. C) The mechanism for CaP‐ink nanoprecipitation and solidification: i) hydrolytic surface degradation of α‐TCP as glycerol is replaced with water; ii) Ca‐P nucleation and growth catalyzed by ammonium phosphate dibasic (APD); iii) polyoxyethylenesorbitan monooleate (PS) directs nanocrystal growth and crystal entanglement. D) Storage modulus (red triangles) and apparent viscosity (blue circles) of ink as a function of time in humid and dry (inset) condition. E) Distribution of bovine serum albumin labeled with fluorescein isothiocyanate (FITC) in cross‐section of scaffolds incorporated by submerging a sintered scaffold in the protein solution (top) or direct mixing of the protein with ink (bottom). F) Comparative drug release profiles of dexamethasone (brown circles) and ibuprofen (blue squares) loaded into CaP‐ink scaffolds and sintered hydroxyapatite scaffolds showing higher controlled release when drugs are incorporated in ink. G) Compressive strength of printed CaP‐ink compared to cancellous bone (orange) and sintered scaffolds (light blue).
Computational modeling of the interaction between CaP‐ink filaments and gelatin microspheres in the support bath to identify conditions for the minimum ink deformation after printing. A) Representative arrangements of ink filaments with a diameter of 600 and 200 µm in gelatin microspheres: straight (90°), inclined (45°), and horizontal (0°) in single‐strand and spiral forms. Deformation maps of ink filaments in the single‐strand form with a diameter of B) 600 µm, C) 200 µm, and D) in spiral‐form with a diameter of 200 µm printed in a bath containing gelatin microspheres with a diameter of 600, 300, and 20 µm. MD = microsphere diameter. E) Experimental validation of gelatin microsphere size on the deformation of ink filaments extruded through nozzles with diameters of 220, 430, and 500 µm. F) Size distribution and optical image of crosslinked gelatin microspheres by glutaraldehyde at fully hydrated state: Optical image (20×) of hydrated microparticles synthesized with span surfactant and histogram of size distribution, scale bar 50 µm (S); Optical image (4×) of hydrated microparticles synthesized under standard conditions (M) with the histogram of size distribution; Optical image (4×) of hydrated microparticles synthesized under slow conditions (L) with the histogram of size distribution. Scale bars 350 µm. G) Rheology of glutaraldehyde treated gelatin microsphere bath: i) Complex viscosity versus shear strain rate and, ii) complex viscosity versus shear stress.
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2008216 (1 of 12)
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Synthetic Bone-Like Structures Through Omnidirectional
Ceramic Bioprinting in Cell Suspensions
Sara Romanazzo, Thomas Gregory Molley, Stephanie Nemec, Kang Lin, Rakib Sheikh,
John Justin Gooding, Boyang Wan, Qing Li, Kristopher Alan Kilian,* and Iman Roohani*
The integration of hierarchical structure, chemistry, and functional activity
within tissue-engineered scaolds is of great importance in mimicking native
bone tissue. Bone is a highly mineralized tissue which forms at ambient con-
ditions by continuous crystallization of the mineral phase within an organic
matrix in the presence of bone residing cells. Despite recent advances in the
biofabrication of complex engineered tissues, replication of the heterogeneity
of bone microenvironments has been a major challenge in constructing bio-
mimetic bone scaolds. Herein, inspired by the bone biomineralization pro-
cess, the first example of bone mimicking constructs by 3D writing of a novel
apatite-transforming ink in a supportive microgel matrix with living cells is
demonstrated.Using this technique, complex bone-mimicked constructs are
made at room temperature without requiring invasive chemicals, radiation, or
postprocessing steps. This study demonstrates that mineralized constructs
can be deposited within a high density of stem cells, directing the cellular
organization, and promoting osteogenesis in vitro. These findings oer a new
strategy for fabrication of bone mimicking constructs for bone tissue regen-
eration with scope to generate custom bone microenvironments for disease
modeling, multicellular delivery, and in vivo bone repair.
DOI: 10.1002/adfm.202008216
1. Introduction
Bone tissue is an essential part of the human body, playing
roles in mechanical support and protection, mineral homeo-
stasis, and hematopoiesis. Over the past years, there have been
many eorts to mimic bone tissue in the form of 3D tissue-
engineered constructs for the regeneration of the damaged
tissue, disease modeling, drug screening, or simply studying
cell–cell crosstalk in the bone microenvironment.[1–6]
Structurally, bone tissue is an organic–inorganic composite
where metabolically active cells are embedded within a highly
The ORCID identification number(s) for the author(s) of this article
can be found under./adfm..
Dr. S. Romanazzo, K. Lin, R. Sheikh, Prof. K. A. Kilian, Dr. I. Roohani
School of Chemistry
Australian Centre for Nanomedicine
University of New South Wales
Sydney, NSW , Australia
T. G. Molley, S. Nemec, Prof. K. A. Kilian
School of Materials Science and Engineering
University of New South Wales
Sydney, NSW , Australia
Prof. J. J. Gooding
School of Chemistry
Australian Centre for NanoMedicine and the ARC Centre of Excellence in
Convergent Bio-Nano Science and Technology
University of New South Wales
Sydney, NSW , Australia
B. Wan, Prof. Q. Li
School of Aerospace, Mechanical and Mechatronic Engineering
University of Sydney
Sydney, NSW , Australia
mineralized matrix in a hierarchical struc-
tural organization.[7] This has posed a
significant challenge in developing a syn-
thetic approach to replicate the heteroge-
neous environment of bone, which allows
creating mechanically-stable mineralized
constructs and at the same time enables
embedding bone relevant cells and other
temperature-chemical-radiation sensitive
biomolecules. In this domain, a plethora
of materials including bioceramics,[8–12]
cell-laden hydrogels, [5,13,14] and synthetic
thermoplastics,[15] in conjunction with
additive manufacturing techniques have
been employed to create synthetic bone
The rapid advances in 3D printing
techniques of bioceramics (such as litho-
graphic printing) have facilitated the fab-
rication of complex bone-mimicked struc-
tures from a range of bioceramic mate-
rials.[9] For example, Zhang et al. have
recently developed Haversian bone-mim-
icked scaolds from Akermanite using
digital laser processing technique.[10] They designed a series
of scaolds with an integrated hierarchical structure including
Haversian canals, Volkmanncanals, and cancellous bone and
showed their favorable osteogenesis and angiogenesis both in
vitro and in vivo. Despite the robustness and precision of recent
3D printing techniques for bioceramics, prints should be ulti-
mately sintered at high temperatures before proceeding to be
seeded with cells or implantation in vivo. The sintering step is
necessary for removing the organic components that are pri-
marily mixed with the ceramic powder and also for solidifying
the structure. In doing that, temperature-sensitive components
Adv. Funct. Mater. 2021, 31, 
... b) (left) represents the simplified nozzle design used for coaxial bioprinting with ceramic as core and bioink as shell, (right) post-processing for the 3DBP scaffold involves immersion in CaCl2 and PBS [81]. c) Depicts the direct-ink-writing reported by Romanazzo et al. towards 3D printing CaP ink in cell-laden support bath, resulting in high celladhesion with 3DP ink and tailored cellular distribution [184]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A c c e p t e d M a n u s c r i p t ...
... Despite significant progress in 3D printed implants, many still involve stacking planar layers, limiting the printable shapes. Romanazzo et al. introduced 'COBICS' (ceramic omnidirectional bioprinting in cell-suspension) for 3D bioprinting freeform, biomimetic orthopedic constructs [184]. Similar to ...
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Rehabilitative capabilities of any tissue engineered scaffold rely primarily on the triad of i) biomechanical properties such as mechanical properties and architecture, ii) chemical behaviour such as regulation of cytokine expression, and iii) cellular response modulation (including their recruitment and differentiation). The closer the implant can mimic the native tissue, the better it can rehabilitate the damage therein. Among the available fabrication techniques, only 3D bioprinting (3DBP) can satisfactorily replicate the inherent heterogeneity of the host tissue. However, 3DBP scaffolds typically suffer from poor mechanical properties, thereby, driving the increased research interest in development of load-bearing 3DBP orthopaedic scaffolds in recent years. Typically, these scaffolds involve multi-material 3D printing, comprising of at-least one bioink and a load-bearing ink; such that mechanical and biological requirements of the biomaterials are decoupled. Ensuring high cellular survivability and good mechanical properties is a key concern in all these studies. 3DBP of such scaffolds is in early developmental stages, and research data from only a handful of preliminary animal studies are available, owing to limitations in print-capabilities and restrictive materials library. This article presents a topically focused review of the state-of-the-art, while highlighting aspects like available 3DBP techniques; biomaterials’ printability; mechanical and degradation behaviour; and their overall bone-tissue rehabilitative efficacy. This collection amalgamates and critically analyses the research aimed at 3DBP of load-bearing scaffolds for fulfilling demands of personalized-medicine. We highlight the recent-advances in 3DBP techniques employing thermoplastics and phosphate-cements for load-bearing applications. Finally, we provide an outlook for possible future perspectives of 3DBP for load-bearing orthopaedic applications. Overall, the article creates ample foundation for future research, as it gathers the latest and ongoing research that scientists could utilize.
... [27,28] However, a supportive gel matrix or a post heat-treatment is usually needed to strengthen the printed structures. [29][30][31][32] Unfortunately, the post-processing step is energy-consuming, especially if sintering is involved, and prevents the use of temperaturesensitive components. It also typically compromises the shape fidelity of the materials as it involves significant shrinkage of the printed parts. ...
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Nature fabricates hard functional materials from soft organic scaffolds that are mineralized. To enable an energy‐efficient locomotion of these creatures while maintaining their structural stability, nature often renders parts of these minerals porous. Unfortunately, methods to produce synthetic minerals with a similar degree of control over their multi length scale porous structure remain elusive. This level of control, however, would be required to design lightweight yet robust biominerals. Here, a room temperature process is presented that combines a localized mineralization with emulsion‐based 3D printing to form cm sized biominerals possessing pores whose diameters range from the 100 s of nm up to the mm length scale. The samples encompass up to 80 wt% of CaCO3 and display a specific compressive strength that is significantly higher than that of previously reported 3D printed porous biominerals and close to those of trabecular bones. The universality of this approach by forming different types of bioactive minerals, including calcite, aragonite, and brushite is demonstrated. The ability to 3D print these materials under benign conditions renders this energy‐efficient process well‐suited to construct cm‐sized lightweight yet load‐bearing structures that might find applications, for example, in the design of the next generation of flying or motile objects.
... Newly developed biomaterials are also opening the door to the possibility of 3D printing directly into patients in the hospitals of the future. A research group from the University of New South Wales developed a cell-based bioink which can be printed via light portable 3D printer and harden within 5-10 min in the presence of water or bodily fluids (Romanazzo et al., 2021;Min, 2021). The properties of the novel bioink and instrument open the door to print directly into the defect site of patients in real time during surgeries after trauma or injury, potentially mitigating the need to reopen a patient for corrective surgery later. ...
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The recent devastating pandemic has drastically reminded humanity of the importance of constant scientific and technological progress. A strong interdisciplinary dialogue between academic and industrial scientists of various specialties, entrepreneurs, managers and the public is paramount in triggering new breakthrough ideas which often emerge at the interface of disciplines. The following sections, compiled by a highly diverse group of authors, are summarizing recently achieved game-changing leaps in science and technology. The game-changers range from paradigm shifts in scientific theories to make impact over several decades to game-changers that have the potential to change our everyday lives tomorrow. The paper is an interdisciplinary dialogue of relevance for academic interdisciplinary thinkers, large corporations' strategic planners, and top executives alike; it provides a glimpse into what further breakthroughs the future may hold and thereby intends to spark new ideas with its readers.
... Since 2018, we have run an internationally attended open-source 3D bioprinting workshop at Carnegie Mellon University where participants build their own bioprinter, learn how to use them for Freeform Reversible Embedding of Suspended Hydrogel (FRESH) 3D bioprinting, and then take the bioprinters back to their home institutions for future research. These efforts have served to validate our bioprinter designs and modifications and step-by-step guides for a range user backgrounds and experience levels, and have produced multiple high-impact publications [21][22][23][24][25][26][27] . While we use a FlashForge Finder as the printer here, the approach we describe is readily adaptable to nearly any low-cost and open-source extrusion 3D printer on the market. ...
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The application of 3D printing to biological research has provided the tissue engineering community with a method for organizing cells and biological materials into complex 3D structures. While many commercial bioprinting platforms exist, they are expensive, ranging from $5000 to over $1,000,000. This high cost of entry prevents many labs from incorporating 3D bioprinting into their research. Due to the open-source nature of desktop plastic 3D printers, an alternative option has been to convert low-cost plastic printers into bioprinters. Several open-source modifications have been described, but there remains a need for a user-friendly, step-by-step guide for converting a thermoplastic printer into a bioprinter using components with validated performance. Here we convert a low-cost 3D printer, the FlashForge Finder, into a bioprinter using our Replistruder 4 syringe pump and the Duet3D Duet 2 WiFi for total cost of less than $900. We demonstrate that the accuracy of the bioprinter’s travel is better than 35 µm in all three axes and quantify fidelity by printing square lattice collagen scaffolds with average errors less than 2%. We also show high fidelity reproduction of clinical-imaging data by printing a scaffold of a human ear using collagen bioink. Finally, to maximize accessibility and customizability, all components we have designed for the bioprinter conversion are provided as open-source 3D models, along with instructions for further modifying the bioprinter for additional use cases, resulting in a comprehensive guide for the bioprinting field.
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If I only had a heart 3D bioprinting is still a fairly new technique that has been limited in terms of resolution and by the materials that can be printed. Lee et al. describe a 3D printing technique to build complex collagen scaffolds for engineering biological tissues (see the Perspective by Dasgupta and Black). Collagen gelation was controlled by modulation of pH and could provide up to 10-micrometer resolution on printing. Cells could be embedded in the collagen or pores could be introduced into the scaffold via embedding of gelatin spheres. The authors demonstrated successful 3D printing of five components of the human heart spanning capillary to full-organ scale, which they validated for tissue and organ function. Science , this issue p. 482 ; see also p. 446
Scaffold-free engineering of three-dimensional (3D) tissue has focused on building sophisticated structures to achieve functional constructs. Although the development of advanced manufacturing techniques such as 3D printing has brought remarkable capabilities to the field of tissue engineering, creating and culturing individual cell-only based high-resolution tissues with complex geometries without an intervening biomaterial scaffold while maintaining the resulting constructs' shape and architecture over time has not been achieved to date. In this report, we introduce a cell printing platform which addresses the aforementioned challenge and permits 3D printing and long-term culture of a living cell-only bioink lacking a biomaterial carrier for functional tissue formation. A biodegradable and photocrosslinkable microgel supporting bath serves initially as a fluid, allowing free movement of the printing nozzle for high-resolution cell extrusion, while also presenting solid-like properties to sustain the structure of the printed constructs. The printed human stem cells, which are the only component of the bioink, couple together via transmembrane adhesion proteins and differentiate down tissue-specific lineages while being cultured in a further photocrosslinked supporting bath to form bone and cartilage tissue with precisely controlled structure. Collectively, this system, which is applicable to general 3D printing strategies, is a paradigm shift for printing of scaffold-free individual cells, cellular condensations and organoids, and may have far reaching impact in the fields of regenerative medicine, drug screening, and developmental biology.