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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, 
... 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.
... [12,13,14] Pioneering studies have demonstrated that jammed microgel assemblies (i.e., granular gels) fulfill these requirements and exhibit excellent rheological properties as supports for embedded printing of delicate structures from soft hydrogels and cells. [15,16] Since then, embedded printing has evolved into a versatile biofabrication platform used for the manufacturing of anatomically accurate tissue components, [17,18,19], [20] patterning of cellular spheroids, [21], [22] engineering of perfusable channels, [23,24,25] and investigation of cellgenerated forces. [26,27] Unfortunately, in contrast to the vast material landscape offered by bulk hydrogels, granular gels designed for embedded printing have been restricted to a handful of materials that allow simple chemical formulation and cost-effective production of microparticles in large quantities (e.g., gelatin, Carbopol, agarose). ...
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Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high‐fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self‐Healing Annealable Particle‐Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular‐matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype‐specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi‐ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular‐like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.
... 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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Advanced additive manufacturing techniques have been recently used to tackle the two fundamental challenges of biodegradable Fe-based bone-substituting materials, namely low rate of biodegradation and insufficient bioactivity. While additively manufactured porous iron has been somewhat successful in addressing the first challenge, the limited bioactivity of these biomaterials hinder their progress towards clinical application. Herein, we used extrusion-based 3D printing for additive manufacturing of iron-matrix composites containing silicate-based bioceramic particles (akermanite), thereby addressing both of the abovementioned challenges. We developed inks that carried iron and 5, 10, 15, or 20 vol% of akermanite powder mixtures for the 3D printing process and optimized the debinding and sintering steps to produce geometrically-ordered iron-akermanite composites with an open porosity of 69–71%. The composite scaffolds preserved the designed geometry and the original α-Fe and akermanite phases. The in vitro biodegradation rates of the composites were improved as much as 2.6 times the biodegradation rate of geometrically identical pure iron. The yield strengths and elastic moduli of the scaffolds remained within the range of the mechanical properties of the cancellous bone, even after 28 days of biodegradation. The composite scaffolds (10–20 vol% akermanite) demonstrated improved MC3T3-E1 cell adhesion and higher levels of cell proliferation. The cellular secretion of collagen type-1 and the alkaline phosphatase activity on the composite scaffolds (10–20 vol% akermanite) were, respectively higher than and comparable to Ti6Al4V in osteogenic medium. Taken together, these results clearly show the potential of 3D printed porous iron-akermanite composites for further development as promising bone substitutes. Statement of significance Porous iron matrix composites containing akermanite particles were produced by means of multi-material additive manufacturing to address the two fundamental challenges associated with biodegradable iron-based biomaterials, namely very low rate of biodegradation and insufficient bioactivity. Our porous iron-akermanite composites exhibited enhanced biodegradability and superior bioactivity compared to porous monolithic iron scaffolds. The murine bone cells proliferated on the composite scaffolds, and secreted the collagen type-1 matrix that stimulated bony-like mineralization. The results show the exceptional potential of the developed porous iron-based composite scaffolds for application as bone substitutes.
... Bone tissue is an essential part of the human body and plays a role in mechanical support and protection, mineral homeostasis, and hematopoiesis (Romanazzo et al., 2021). Over the past few years, many efforts have been made to use 3D printing technology to regenerate damaged bone tissue. ...
Full-text available
As a microenvironment where cells reside, the extracellular matrix (ECM) has a complex network structure and appropriate mechanical properties to provide structural and biochemical support for the surrounding cells. In tissue engineering, the ECM and its derivatives can mitigate foreign body responses by presenting ECM molecules at the interface between materials and tissues. With the widespread application of three-dimensional (3D) bioprinting, the use of the ECM and its derivative bioinks for 3D bioprinting to replicate biomimetic and complex tissue structures has become an innovative and successful strategy in medical fields. In this review, we summarize the significance and recent progress of ECM-based biomaterials in 3D bioprinting. Then, we discuss the most relevant applications of ECM-based biomaterials in 3D bioprinting, such as tissue regeneration and cancer research. Furthermore, we present the status of ECM-based biomaterials in current research and discuss future development prospects.
... 3D bioprinting, a form of 3DP, focuses on the organized deposition of biological substances (bioinks) and cells [179,180] with several key advantages over 3DP of non-biological substances. These include the ability to directly incorporate cells during the printing process [181,182], 3DP of discrete biological compartments using support baths [183,184] and the implementation of in-situ 3DP of constructs [185,186]. The ability to bioprint craniofacial structures is currently the subject of exciting preclinical studies [187,188]. ...
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Compared to traditional manufacturing methods, additive manufacturing and 3D printing stand out in their ability to rapidly fabricate complex structures and precise geometries. The growing need for products with different designs, purposes and materials led to the development of 3D printing, serving as a driving force for the 4th industrial revolution and digitization of manufacturing. 3D printing has had a global impact on healthcare, with patient-customized implants now replacing generic implantable medical devices. This revolution has had a particularly significant impact on oral and maxillofacial surgery, where surgeons rely on precision medicine in everyday practice. Trauma, orthognathic surgery and total joint replacement therapy represent several examples of treatments improved by 3D technologies. The widespread and rapid implementation of 3D technologies in clinical settings has led to the development of point-of-care treatment facilities with in-house infrastructure, enabling surgical teams to participate in the 3D design and manufacturing of devices. 3D technologies have had a tremendous impact on clinical outcomes and on the way clinicians approach treatment planning. The current review offers our perspective on the implementation of 3D-based technologies in the field of oral and maxillofacial surgery, while indicating major clinical applications. Moreover, the current report outlines the 3D printing point-of-care concept in the field of oral and maxillofacial surgery.
... mm (Lim et al., 2018a;Muralidharan et al., 2019) 0.037 mm (Peele et al., 2015) 0.02-0.05 mm (Lim et al., 2018b;Wang et al., 2015;Zhang et al., 2021b) Extrusion-based printing 0.1-1.5 mm (Chung et al., 2021;Kajave et al., 2021;Murphy et al., 2017;Romanazzo et al., 2021;Sears et al., 2017) 0.21-0.3 mm (Choi et al., 2019;Laternser et al., 2018) 0.33-0.5 mm (Chae et al., 2021;Laternser et al., 2018;Pati et al., 2014b) Selective laser sintering 0.04-0.45 ...
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This review presents bioprinting methods, biomaterials, and printing strategies that may be used for composite tissue constructs for musculoskeletal applications. The printing methods discussed include those that are suitable for acellular and cellular components, and the biomaterials include soft and rigid components that are suitable for soft and/or hard tissues. We also present strategies that focus on the integration of cell-laden soft and acellular rigid components under a single printing platform. Given the structural and functional complexity of native musculoskeletal tissue, we envision that hybrid bioprinting, referred to as hybprinting, could provide unprecedented potential by combining different materials and bioprinting techniques to engineer and assemble modular tissues.
... 3D printing for prosthetics Another research group has developed a ceramic ink that can be 3D-printed at room temperature with live cells devoid of chemicals [53]. The current gold standard for repairing bone is an autologous bone graft but a high rate of infection is an impediment to this process. ...
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Ageing population and new diseases are requiring the development of novel therapeutical strategies. 3D bioprinting an novel application domain of additive manufacturing emerged as a potential transformative strategy for tissue engineering and regenerative medicine. This paper introduces the concept of 3D bioprinting, discussing in detail key requirements of bio-inks and main materials used to encapsulate cells. Recent advances related to the use of smart materials and the concept of 4D printing is also discussed. Main 3D bioprinting techniques are described in detail and key limitations highlighted. Successful cases, demonstrating the relevance of 3D bioprinting are also presented. Finally, the paper addresses the main research challenges and future perspectives in the field of 3D bioprinting.
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Reconstruction of patient-specific scaffolds to repair uniquely shaped bone defects remains a major clinical challenge in tissue engineering. Recently, three-dimensional (3D) printed scaffolds have received considerable attention as a promising technology for the rapid generation of custom shapes. However, synthetic polymers commonly used for 3D printing, such as polycaprolactone (PCL), lack the biological capacity to mimic native extracellular matrix functions to support cell growth and differentiation into desired tissues. We described the preparation and characterization of a 3D hybrid model for bone tissue engineering that comprises an extracellular matrix (ECM)-enriched hydrogel embedded in a PCL scaffold. The human bone marrow-derived mesenchymal stem cell–derived matrisome (BMTS) was utilized as a source of ECM-enriched biomacromolecules, and scaffold biocompatibility was evaluated in vitro using human bone marrow-derived mesenchymal stem cells (BM-MSCs). The 3D hybrid model exhibited excellent BM-MSC viability and osteogenic activity in vitro in both two-dimensional (2D) and 3D cultures. Furthermore, bone remodeling was evaluated by in vivo through a rat calvarial defect model; notably, the fabricated 3D hybrid model effectively enhanced vascularized bone regeneration. Therefore, this promising BMTS-based 3D hybrid model might serve as an excellent bone tissue-engineered scaffold for use in orthopedic applications.
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The integration of structure and function for tissue engineering scaffolds is of great importance in mimicking native bone tissue. However, the complexity of hierarchical structures, the requirement for mechanical properties, and the diversity of bone resident cells are the major challenges in constructing biomimetic bone tissue engineering scaffolds. Herein, a Haversian bone–mimicking scaffold with integrated hierarchical Haversian bone structure was successfully prepared via digital laser processing (DLP)–based 3D printing. The compressive strength and porosity of scaffolds could be well controlled by altering the parameters of the Haversian bone–mimicking structure. The Haversian bone–mimicking scaffolds showed great potential for multicellular delivery by inducing osteogenic, angiogenic, and neurogenic differentiation in vitro and accelerated the ingrowth of blood vessels and new bone formation in vivo. The work offers a new strategy for designing structured and functionalized biomaterials through mimicking native complex bone tissue for tissue regeneration.
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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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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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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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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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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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Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro–computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.
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.