Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in digital light projection(DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks and model cells. A multiphase many-body dissipative particle dynamics model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4-3 kPa) with a typical layer thickness of 50µm, an XY resolution of 38 ± 1.5µm and Z resolution of 237 ± 5.4µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel networks lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in Digital Light Processing (DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks. A multiphase many-body dissipative particle dynamics (mDPD) model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4 to 3 kPa) with an XY resolution of 38 ± 1.5 µm and Z resolution of 237±5.4 µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel network lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Printing Double-Network Tough Hydrogels Using Temperature-Controlled Projection Stereolithography (TOPS).

We report a new method to shape double-network (DN) hydrogels into customized 3D structures that exhibit superior mechanical properties in both tension and compression. A one-pot prepolymer formulation containing photo-cross-linkable acrylamide and thermoreversible sol-gel κ-carrageenan with a suitable cross-linker and photoinitiators/absorbers is optimized. A new TOPS system is utilized to photopolymerize the primary acrylamide network into a 3D structure above the sol-gel transition of κ-carrageenan (80 °C), while cooling down generates the secondary physical κ-carrageenan network to realize tough DN hydrogel structures. 3D structures, printed with high lateral (37 µm) and vertical (180 µm) resolutions and superior 3D design freedoms (internal voids), exhibit ultimate stress and strain of 200 kPa and 2400%, respectively, under tension and simultaneously exhibit a high compression stress of 15 MPa with a strain of 95%, both with high recovery rates. The roles of swelling, necking, self-healing, cyclic loading, dehydration, and rehydration on the mechanical properties of printed structures are also investigated. To demonstrate the potential of this technology to make mechanically reconfigurable flexible devices, we print an axicon lens and show that a Bessel beam can be dynamically tuned via user-defined tensile stretching of the device. This technique can be broadly applied to other hydrogels to make novel smart multifunctional devices for a range of applications.

Printing Parameters of Fused Filament Fabrication Affect Key Properties of Four-Dimensional Printed Shape-Memory Polymers.

Extrusion-based (fused filament fabrication) three-dimensional (3D) printing of shape-memory polymers (SMPs) has the potential to rapidly produce highly customized smart-material parts. Yet, the effects of printing parameters on the shape-memory properties of printed SMPs remain poorly understood. To study the extent to which the 3D printing process affects the shape-memory properties of a printed SMP part, here temperature, extrusion rate multiplier, and fiber orientation were systematically varied, and their effect on shape-memory fixing and recovery ratios was evaluated. Fiber orientation, as determined by print path relative to the direction(s) of loading during shape-memory programming, was found to significantly impact the fixing ratio and the recovery ratio. Temperature and multiplier had little effect on either fixing ratio or recovery ratio. To facilitate the use of printed SMP parts in biomedical applications, a cell viability assay was performed on 3D-printed samples prepared using varied temperature and multiplier. Reduction in multiplier was found to increase cell viability. The results indicate that fiber orientation can critically impact the shape-memory functionality of 3D-printed SMP parts, and that multiplier can affect cytocompatibility of those parts. Thus, researchers and manufacturers employing SMPs in 3D-printed parts and devices could achieve improved part functionality if print paths are designed to align fiber direction with the axis(es) in which strain will be programmed and recovered and if the multiplier is optimized in biomedical applications in which a part will contact cells.

In vitromodel to study confined osteocyte networks exposed to flow-induced mechanical stimuli.

Osteocytes are considered the primary mechanical sensor in bone tissue and orchestrate the coupled bone remodeling activity of adjacent osteoblast and osteoclast cells.In vivoinvestigation of mechanically induced signal propagation through networks of interconnected osteocytes is confounded by their confinement within the mineralized bone matrix, which cannot be modeled in conventional culture systems. In this study, we developed a new model that mimics thisin vivoconfinement using gelatin methacrylate (GelMA) hydrogel or GelMA mineralized using osteoblast-like model cells. This model also enables real-time optical examination of osteocyte calcium (Ca2+) signaling dynamics in response to fluid shear stimuli cultured under confined conditions. Using this system, we discovered several distinct and previously undescribed patterns of Ca2+responses that vary across networks of interconnected osteocytes as a function of space, time and connectivity. Heterogeneity in Ca2+signaling may provide new insights into bone remodeling in response to mechanical loading. Overall, such a model can be extended to study signaling dynamics within cell networks exposed to flow-induced mechanical stimuli under confined conditions.

Femtosecond Laser Densification of Hydrogels to Generate Customized Volume Diffractive Gratings.

Inspired by nature's ability to shape soft biological materials to exhibit a range of optical functionalities, we report femtosecond (fs) laser-induced densification as a new method to generate volume or subsurface diffractive gratings within ordinary hydrogel materials. We characterize the processing range in terms of fs laser power, speed, and penetration depths for achieving densification within poly(ethylene glycol) diacrylate (PEGDA) hydrogel and characterize the associated change in local refractive index (RI). The RI change facilitates the fabrication of custom volume gratings (parallel line, grid, square, and ring gratings) within PEGDA. To demonstrate this method's broad applicability, fs laser densification was used to generate line gratings within the phenylboronic acid (PBA) hydrogel, which is known to be responsive to changes in pH. In the future, this technique can be used to convert ordinary hydrogels into multicomponent biophotonic systems.

Smart biomaterial platforms: Controlling and being controlled by cells.

Across diverse research and application areas, dynamic functionality-such as programmable changes in biochemical property, in mechanical property, or in microscopic or macroscopic architecture-is an increasingly common biomaterials design criterion, joining long-studied criteria such as cytocompatibility and biocompatibility, drug release kinetics, and controlled degradability or long-term stability in vivo. Despite tremendous effort, achieving dynamic functionality while simultaneously maintaining other desired design criteria remains a significant challenge. Reversible dynamic functionality, rather than one-time or one-way dynamic functionality, is of particular interest but has proven especially challenging. Such reversible functionality could enable studies that address the current gap between the dynamic nature of in vivo biological and biomechanical processes, such as cell traction, cell-extracellular matrix (ECM) interactions, and cell-mediated ECM remodeling, and the static nature of the substrates and ECM constructs used to study the processes. This review assesses dynamic materials that have traditionally been used to control cell activity and static biomaterial constructs, experimental and computational techniques, with features that may inform continued advances in reversible dynamic materials. Taken together, this review presents a perspective on combining the reversibility of smart materials and the in-depth dynamic cell behavior probed by static polymers to design smart bi-directional ECM platforms that can reversibly and repeatedly communicate with cells.

Three-Dimensional Printing of Double-Network Hydrogels: Recent Progress, Challenges, and Future Outlook.

Hydrogels are widely used materials due to their biocompatibility, their ability to mimic the hydrated and porous extracellular microenvironment, as well as their ability to tune both mechanical and biochemical properties. However, most hydrogels lack mechanical toughness, and shaping them into complicated three-dimensional (3D) structures remains challenging. In the past decade, tough and stretchable double-network hydrogels (DN gels) were developed for tissue engineering, soft robotics, and applications that require a combination of high-energy dissipation and large deformations. Although DN gels were processed into simple shapes by using conventional casting and molding methods, new 3D printing methods have enabled the shaping of DN gels into structurally complex 3D geometries. This review will describe the state-of-art technologies for shaping tough and stretchable DN gels into custom geometries by using conventional molding and casting, extrusion, and optics-based 3D printing, as well as the key challenges and future outlook in this field.

Hydrogel-based diffractive optical elements (hDOEs) using rapid digital photopatterning.

Hydrogels, due to their optical transparency and biocompatibility, have emerged as an excellent alternative to conventional optical materials for biomedical applications. Advances in microfabrication techniques have helped convert conventional hydrogels into optically functional materials such as hydrogel-based diffraction optical elements (hDOEs). However, key challenges related to device customization and ease/speed of fabrication need to be addressed to enable widespread utility and acceptance of hDOEs in the field. Here, we report rapid printing of customized hDOEs on polyethylene glycol diacrylate (PEGDA) hydrogel using digital photopatterning; a novel method that combines simulated computer-generated hologram (SCGH) and projection photolithography. To showcase the versatility of this approach, a range of hDOEs are demonstrated, including 1D/2D diffraction gratings, Dammann grating, Fresnel zone plate (FZP) lens, fork-shaped grating and computer-generated hologram (CGH) of arbitrary pattern. Results demonstrate that printed hDOEs exhibit optical performance that is comparable with devices made with conventional materials. This versatile strategy can be potentially implemented with other photosensitive hydrogels to achieve user-defined hDOEs in a time-efficient and cost-effective fashion.

Direct Laser Writing of Fluorescent Silver Nanoclusters: A Review of Methods and Applications.

Metal nanoclusters (NCs) are nanomaterials of size of less than 2 nm that exhibit a set of unique physical, chemical, optical, and electronic properties. Because of recent interest in NCs, a great deal of effort is being made to develop synthetic routes that allow control over the NC size, shape, geometry, and properties. Direct laser writing is one of the few synthesis methods that allow the generation of photostable NCs with high quantum yield in a highly controlled fashion. A key advantage of laser-written NCs is the ability to create easy-to-use solid-state devices for a range of applications. This review will present necessary background and recent advances in laser writing of silver NCs and their applications in different solid-state matrixes such as glass, zeolites, and polymer substrate. This topic will be of interest to researchers in the fields of materials science, optics and photonics, chemistry, and biomedical sciences.

Perfusion-based co-culture model system for bone tissue engineering.

In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.

Oxygen-Permeable Films for Continuous Additive, Subtractive, and Hybrid Additive/Subtractive Manufacturing.

In the past 5 years, oxygen-permeable films have been widely used for continuous additive manufacturing. These films create a polymerization inhibition zone that facilitates continuous printing in the additive mode of fabrication. Typically, oxygen-permeable films made out of Teflon are currently used. These films are expensive and are not commonly available. Hence, this research work investigates the feasibility of using commonly available low-cost oxygen-permeable films made from polydimethylsiloxane (PDMS) and polyurethane for continuous additive manufacturing. We also characterize the ablation depth range that can be achieved using these films and the potential use for subtractive ablation-based manufacturing as well as hybrid additive/subtractive manufacturing. Results demonstrate that the PDMS films (600 µm thick) can be used for both additive and subtractive modes, whereas spin-coated PDMS thin film (40 µm thick) on glass coverslip and breathe-easy polyurethane film (20 µm thick) laminated on glass coverslip are suitable only for additive mode of fabrication. The latter two films are oxygen impermeable, however, they retain oxygen, which is capable of creating dead zone and thereby facilitates continuous printing. We anticipate that this work will help researchers to choose the appropriate oxygen-permeable film for continuous additive, subtractive, and hybrid additive/subtractive manufacturing of complex three-dimensional structures for a range of applications.

High-Resolution 3D Printing of Stretchable Hydrogel Structures Using Optical Projection Lithography.

Double-network (DN) hydrogels, with their unique combination of mechanical strength and toughness, have emerged as promising materials for soft robotics and tissue engineering. In the past decade, significant effort has been devoted to synthesizing DN hydrogels with high stretchability and toughness; however, shaping the DN hydrogels into complex and often necessary user-defined two-dimensional (2D) and three-dimensional (3D) geometries remains a fabrication challenge. Here, we report a new fabrication method based on optical projection lithography to print DN hydrogels into customizable 2D and 3D structures within minutes. DN hydrogels were printed by first photo-crosslinking a single network structure via spatially modulated light patterns followed by immersing the printed structure in a calcium bath to induce ionic cross-linking. Results show that DN structures made by this method can stretch four times their original lengths. We show that strain and the elastic modulus of printed structures can be tuned based on the hydrogel composition, cross-linker and photoinitiator concentrations, and laser light intensity. To our knowledge, this is the first report demonstrating quick lithography and high-resolution printing of DN (covalent and ionic) hydrogels within minutes. The ability to shape tough and stretchable DN hydrogels in complex structures will be potentially useful in a broad range of applications.

A bioink blend for rotary 3D bioprinting tissue engineered small-diameter vascular constructs.

3D bioprinted vascular constructs have gained increased interest due to their significant potential for creating customizable alternatives to autologous vessel grafts. In this study, we developed a new approach for biofabricating fibrin-based vascular constructs using a novel rotary 3D bioprinter developed in our lab. We formulated a new bioink by incorporating fibrinogen with gelatin to achieve a desired shear-thinning property for rotary bioprinting. The blending of heat-treated gelatin with fibrinogen turned unprintable fibrinogen into a printable biomaterial for vessel bioprinting by leveraging the favorable rheological properties of gelatin. We discovered that the heat-treatment of gelatin remarkably affects the rheological properties of a gelatin-fibrinogen blended bioink, which in turn influences the printability of the ink. Further characterizations revealed that not only concentration of the gelatin but the heat treatment also affects cell viability during printing. Notably, the density of cells included in the bioinks also influenced printability and tissue volumetric changes of the printed vessel constructs during cultures. We observed increased collagen deposition and construct mechanical strength during two months of the cultures. The burst pressure of the vessel constructs reached 1110 mmHg, which is about 52% of the value of the human saphenous vein. An analysis of the tensile mechanical properties of the printed vessel constructs unveiled an increase in both the circumferential and axial elastic moduli during cultures. This study highlights important considerations for bioink formulation when bioprinting vessel constructs. STATEMENT OF SIGNIFICANCE: There has been an increased demand for small-diameter tissue-engineered vascular grafts. Vascular 3D bioprinting holds the potential to create equivalent vascular grafts but with the ability to tailor them to meet patient's needs. Here, we presented a new and innovative 3D rotary bioprinter and a new bioink formulation for printing vascular constructs using fibrinogen, a favorable biomaterial for vascular tissue engineering. The bioink was formulated by blending fibrinogen with a more printable biomaterial, gelatin. The systematic characterization of the effects of heat treatment and gelatin concentration as well as bioink cell concentration on the printability of the bioink offers new insight into the development of printable biomaterials for tissue biofabrication.

Femtosecond laser induced densification within cell-laden hydrogels results in cellular alignment.

The unique capabilities of ultrafast lasers to introduce user-defined microscale modifications within 3D cell-laden hydrogels have been used to investigate fundamental cellular phenomenon such as adhesion, alignment, migration and organization. In this work, we report a new material modification phenomenon coined as 'densification' and its influence on the behavior of encapsulated cells. Femtosecond laser writing technique was used to write densified lines of width 1-5 µm within the bulk of gelatin methacrylate (GelMA) constructs. We found that densified micro-lines within cell-laden GelMA constructs resulted in preferential and localized alignment of encapsulated human endothelial cells. Degree of cellular alignment was characterized as a function of cell-culture time and the spacing between the densified line patterns. This phenomenon was found to be true for several cell lines, including mouse fibroblasts and osteocytes, and mesenchymal stem cells derived from human induced pluripotent cells. This first report of physical densification using fs lasers can be potentially extended for investigating cell behavior within other photosensitive hydrogels.

Serum-Free Manufacturing of Mesenchymal Stem Cell Tissue Rings Using Human-Induced Pluripotent Stem Cells.

Combination of stem cell technology and 3D biofabrication approaches provides physiological similarity to in vivo tissues and the capability of repairing and regenerating damaged human tissues. Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine applications because of their immunosuppressive properties and multipotent potentials. To obtain large amount of high-quality MSCs without patient donation and invasive procedures, we differentiated MSCs from human-induced pluripotent stem cells (hiPSC-MSCs) using serum-free E6 media supplemented with only one growth factor (bFGF) and two small molecules (SB431542 and CHIR99021). The differentiated cells showed a high expression of common MSC-specific surface markers (CD90, CD73, CD105, CD106, CD146, and CD166) and a high potency for osteogenic and chondrogenic differentiation. With these cells, we have been able to manufacture MSC tissue rings with high consistency and robustness in pluronic-coated reusable PDMS devices. The MSC tissue rings were characterized based on inner diameter and outer ring diameter and observed cell-type-dependent tissue contraction induced by cell-matrix interaction. Our approach of simplified hiPSC-MSC differentiation, modular fabrication procedure, and serum-free culture conditions has a great potential for scalable manufacturing of MSC tissue rings for different regenerative medicine applications.

Hybrid Laser Printing of 3D, Multiscale, Multimaterial Hydrogel Structures.

Fabrication of multiscale, multi-material three-dimensional (3D) structures at high resolution is difficult using current technologies. This is especially significant when working with hydrated and mechanically weak hydrogel materials. In this work, a new hybrid laser printing (HLP) technology is reported to print complex, multiscale, multimaterial, 3D hydrogel structures with microscale resolution. This technique of fabrication utilizes sequential additive and subtractive modes of material fabrication, that are typically considered as mutually exclusive due to differences in their material processing conditions. Further, compared to current laser writing systems that enforce stringent processing depth limits, HLP is shown to fabricate structures at any depth inside the material. As a proof-of-principle, a Mayan Pyramid with embedded cube-frame is printed using model synthetic polyethylene glycol diacrylate (PEGDA) hydrogel. Printing of ready-to-use open-well chips with embedded microchannels is also demonstrated using PEGDA and gelatin methacrylate (GelMA) hydrogels for potential applications in biomedical sciences. Next, HLP is used in additive and additive modes to print multiscale 3D structures spanning in size from centimeter to micrometers within minutes, which is followed by printing of 3D, multi-material, multiscale structures using this technology. Overall, this work demonstrates that HLP's fabrication versatility can potentially offer a unique opportunity for a range of applications in optics and photonics, biomedical sciences, microfluidics, soft robotics, etc.

Measuring Changes in Electrical Impedance During Cell-Mediated Mineralization.

Background: The fundamental electrical properties of bone have been attributed to the organic collagen and the inorganic mineral component; however, contributions of individual components within bone tissue toward the measured electrical properties are not known. In our study, we investigated the electrical properties of cell-mediated mineral deposition process and compared our results with cell-free mineralization. Materials and Methods: Saos-2 cells encapsulated within gelatin methacrylate (GelMA) hydrogels were chemically stimulated in osteogenic medium for a period of 4 weeks. The morphology, composition, and mechanical properties of the mineralized constructs were characterized using bright-field imaging, scanning electron microscopy (SEM) energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy (FITR), nuclear magnetic resonance spectroscopy (NMR), micro-CT, immunostaining, and mechanical compression tests. In parallel, a custom-made device was used to measure the electrical impedance of mineralized constructs. All results were compared with cell-free GelMA hydrogels mineralized through the simulated body fluid approach. Results: Results demonstrate a decrease in the electrical impedance of deposited mineral in both cell-mineralized and cell-free mineralized samples. Conclusions: This study establishes a model system to investigate in vivo and in vitro mineralization processes.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via micro-CT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. Micro-CT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels.