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

DOI:10.1002/adfm.202008216
Authors:
Sara Romanazzo at UNSW Sydney
Sara Romanazzo
Thomas Gregory Molley
Thomas Gregory Molley
Thomas Gregory Molley
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Stephanie Nemec
Stephanie Nemec
Stephanie Nemec
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Kang Lin at UNSW Sydney
Kang Lin
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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.
Overview of the process of omnidirectional printing of highly mineralized bone mimicked constructs under mild conditions and in the presence of live cells.
Overview of the process of omnidirectional printing of highly mineralized bone mimicked constructs under mild conditions and in the presence of live cells.
… 
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).
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.
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.
… 
Generation of complex bone‐mimetic architectures in a one‐step procedure. A) Photographs of a printed CaP‐ink filament within a gelatin microsphere support bath and interfacial adhesion during nanocrystal formation; B) front view of the printing process in a 96‐well cell culture plate. Example of 3D constructs printed by COBICS: C) a heterogeneous biphasic structure mimicking osteochondral tissue using CaP‐ink, gelatin microspheres and agarose hydrogel, and bone‐like structures, such as D) human osseous labyrinth, E) trabecular bone, and F) helical Haversian canal. Magnification of printed BSA‐FITC‐labeled ink observed at epifluorescence microscope (F, middle) and at SEM to show formation of hydroxyapatite crystals postprinting (F, right).
Generation of complex bone‐mimetic architectures in a one‐step procedure. A) Photographs of a printed CaP‐ink filament within a gelatin microsphere support bath and interfacial adhesion during nanocrystal formation; B) front view of the printing process in a 96‐well cell culture plate. Example of 3D constructs printed by COBICS: C) a heterogeneous biphasic structure mimicking osteochondral tissue using CaP‐ink, gelatin microspheres and agarose hydrogel, and bone‐like structures, such as D) human osseous labyrinth, E) trabecular bone, and F) helical Haversian canal. Magnification of printed BSA‐FITC‐labeled ink observed at epifluorescence microscope (F, middle) and at SEM to show formation of hydroxyapatite crystals postprinting (F, right).
… 
Cytocompatibility of bone‐mimetic constructs. A) viability staining of mesenchymal stem cells (MSC), including bone‐marrow MSC (BM‐MSC) and adipose‐derived MSC (ADSC), and osteoblast cells seeded on CaP‐ink and scanning electron microscopy of MSC on CaP‐ink after 7 days, showing the production of mineral nodules indicating the transition of MSC toward osteoblast. B) Top view of 3D printed construct after 7 days culture with mesenchymal stem cells (MSCs), showing tissue formation around the ink (left) and live‐dead images of day 14 cell culture in COBICS (middle and right). C) Cell viability and cell shape analysis of MSC cultured on ceramic ink i,ii), cell viability of human osteoblasts on ink iii), MSC viability embedded within the support bath and migration of MSC toward CaP‐ink iv).
+1
Cytocompatibility of bone‐mimetic constructs. A) viability staining of mesenchymal stem cells (MSC), including bone‐marrow MSC (BM‐MSC) and adipose‐derived MSC (ADSC), and osteoblast cells seeded on CaP‐ink and scanning electron microscopy of MSC on CaP‐ink after 7 days, showing the production of mineral nodules indicating the transition of MSC toward osteoblast. B) Top view of 3D printed construct after 7 days culture with mesenchymal stem cells (MSCs), showing tissue formation around the ink (left) and live‐dead images of day 14 cell culture in COBICS (middle and right). C) Cell viability and cell shape analysis of MSC cultured on ceramic ink i,ii), cell viability of human osteoblasts on ink iii), MSC viability embedded within the support bath and migration of MSC toward CaP‐ink iv).
… 
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2008216 (1 of 12)
Full PaPer
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 https://doi.org/./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
E-mail: k.kilian@unsw.edu.au; iman.roohani@unsw.edu.au
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
matrices.
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 ...
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... [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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  • Jan 2020
Tissue engineering is promising in realizing successful treatments of human body tissue loss that current methods cannot treat well or achieve satisfactory clinical outcomes. In scaffold-based bone tissue engineering, a high performance scaffold underpins the success of a bone tissue engineering strategy and a major direction in the field is to produce bone tissue engineering scaffolds with desirable shape, structural, physical, chemical and biological features for enhanced biological performance and for regenerating complex bone tissues. Three-dimensional (3D) printing can produce customized scaffolds that are highly desirable for bone tissue engineering. The enormous interest in 3D printing and 3D printed objects by the science, engineering and medical communities has led to various developments of the 3D printing technology and wide investigations of 3D printed products in many industries, including biomedical engineering, over the past decade. It is now possible to create novel bone tissue engineering scaffolds with customized shape, architecture, favorable macro-micro structure, wettability, mechanical strength and cellular responses. This article provides a concise review of recent advances in the R & D of 3D printing of bone tissue engineering scaffolds. It also presents our philosophy and research in the designing and fabrication of bone tissue engineering scaffolds through 3D printing.
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Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces
Article
Full-text available
  • Feb 2020
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-tricalciumphosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled 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 vs. 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.
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3D Printing of Bioceramic Scaffolds—Barriers to the Clinical Translation: From Promise to Reality, and Future Perspectives
Article
Full-text available
  • Aug 2019
In this review, we summarize the challenges of the three-dimensional (3D) printing of porous bioceramics and their translational hurdles to clinical applications. The state-of-the-art of the major 3D printing techniques (powder-based and slurry-based), their limitations and key processing parameters are discussed in detail. The significant roadblocks that prevent implementation of 3D printed bioceramics in tissue engineering strategies, and medical applications are outlined, and the future directions where new research may overcome the limitations are proposed. In recent years, there has been an increasing demand for a nanoscale control in 3D fabrication of bioceramic scaffolds via emerging techniques such as digital light processing, two-photon polymerization, or large area maskless photopolymerization. However, these techniques are still in a developmental stage and not capable of fabrication of large-sized bioceramic scaffolds; thus, there is a lack of sufficient data to evaluate their contribution. This review will also not cover polymer matrix composites reinforced with particulate bioceramics, hydrogels reinforced with particulate bioceramics, polymers coated with bioceramics and non-porous bioceramics.
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Volumetric Bioprinting of Complex Living‐Tissue Constructs within Seconds
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Biofabrication technologies, including stereolithography and extrusion-based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer-by-layer deposition and assembly of repetitive building blocks, typically cell-laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical-tomography-inspired printing approach, based on visible light projection, is developed to generate cell-laden tissue constructs with high viability (>85%) from gelatin-based photoresponsive hydrogels. Free-form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo-fibrocartilage matrix. Moreover, free-floating structures are generated, as demonstrated by printing functional hydrogel-based ball-and-cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter-scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel-based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.
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Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization
Article
Full-text available
  • Aug 2019
Bone tissue, by definition, is an organic-inorganic nanocomposite, where metabolically active cells are embedded within a matrix that is heavily calcified on the nanoscale. Currently, there are no strategies that replicate these definitive characteristics of bone tissue. Here we describe a biomimetic approach where a supersaturated calcium and phosphate medium is used in combination with a non-collagenous protein analog to direct the deposition of nanoscale apatite, both in the intra- and extrafibrillar spaces of collagen embedded with osteoprogenitor, vascular, and neural cells. This process enables engineering of bone models replicating the key hallmarks of the bone cellular and extracellular microenvironment, including its protein-guided biomineralization, nanostructure, vasculature, innervation, inherent osteoinductive properties (without exogenous supplements), and cell-homing effects on bone-targeting diseases, such as prostate cancer. Ultimately, this approach enables fabrication of bone-like tissue models with high levels of biomimicry that may have broad implications for disease modeling, drug discovery, and regenerative engineering.
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3D bioprinting of collagen to rebuild components of the human heart
Article
Full-text available
  • Aug 2019
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
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Individual cell-only bioink and photocurable supporting medium for 3D printing and generation of engineered tissues with complex geometries
Article
  • Jun 2019
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.
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