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

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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).
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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 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, 
... In research conducted at the University of New South Wales in Australia, a scientific team was able to 3D print bone parts with living cells with multidirectional ceramic bioprinting, enabling them to repair damaged bone tissue that hardens within minutes when placed in water. It is a pioneering experiment and the first time that such materials can be created at room temperature with living cells and without harsh chemicals or radiation [71]. This study demonstrates that within a high density of stem cells, directed by cellular regulation, osteogenesis could be promoted in vitro. ...
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Trauma and bone loss from infections, tumors, and congenital diseases make bone repair and regeneration the greatest challenges in orthopedic, craniofacial, and plastic surgeries. The shortage of donors, intrinsic limitations, and complications in transplantation have led to more focus and interest in regenerative medicine. Structures that closely mimic bone tissue can be produced by this unique technology. The steady development of three-dimensional (3D)-printed bone tissue engineering scaffold therapy has played an important role in achieving the desired goal. Bioceramic scaffolds are widely studied and appear to be the most promising solution. In addition, 3D printing technology can simulate mechanical and biological surface properties and print with high precision complex internal and external structures to match their functional properties. Inkjet, extrusion, and light-based 3D printing are among the rapidly advancing bone bioprinting technologies. Furthermore, stem cell therapy has recently shown an important role in this field, although large tissue defects are difficult to fill by injection alone. The combination of 3D-printed bone tissue engineering scaffolds with stem cells has shown very promising results. Therefore, biocompatible artificial tissue engineering with living cells is the key element required for clinical applications where there is a high demand for bone defect repair. Furthermore, the emergence of various advanced manufacturing technologies has made the form of biomaterials and their functions, composition, and structure more diversified, and manifold. The importance of this article lies in that it aims to briefly review the main principles and characteristics of the currently available methods in orthopedic bioprinting technology to prepare bioceramic scaffolds, and finally discuss the challenges and prospects for applications in this promising and vital field.
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... defects [1][2][3][4] . To aid in the regeneration of bone tissue, the biomaterials should (i) fit the anatomical site of the bone defect [5][6][7] , (ii) have mechanical properties in the range of those of bone tissue to preclude the stress shielding effect [8][9][10] , (iii) possess a high degree of interconnected porosity to facilitate angiogenesis, bone ingrowth, as well as the transport of nutrients [11][12][13] , and (iv) biodegrade in vivo while providing the required level of mechanical support during the entire process of bone tissue regeneration [14][15][16] . To date, the low rate of in vivo biodegradation and the inadequate level of osteogenic properties have been the main hinderances preventing Fe-based bone substitutes from widespread clinical application [17] . ...
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