Development of a patient specific cartilage graft using magnetic resonance imaging and 3D printing.

OBJECTIVES: The goal of this project was to develop and validate a patient-specific, anatomically correct graft for cartilage restoration using magnetic resonance imaging (MRI) data and 3-dimensional (3D) printing technology. The specific aim was to test the accuracy of a novel method for 3D printing and implanting individualized, anatomically shaped bio-scaffolds to treat cartilage defects in a human cadaveric model. We hypothesized that an individualized, anatomic 3D-printed scaffold designed from MRI data would provide a more optimal fill for a large cartilage defect compared to a generic flat scaffold. METHODS: Four focal cartilage defects (FCDs) were created in paired human cadaver knees, age <40 years, in the weight-bearing surfaces of the medial femoral condyle (MFC), lateral femoral condyle (LFC), patella, and trochlea of each knee. MRIs were obtained, anatomic grafts were designed and 3D printed for the left knee as an experimental group, and generic flat grafts for the right knee as a control group. Grafts were implanted into corresponding defects and fixed using tissue adhesive. Repeat post-implant MRIs were obtained. Graft step-off was measured as the distance in mm between the surface of the graft and the native cartilage surface in a direction perpendicular to the subchondral bone. Graft contour was measured as the gap between the undersurface of the graft and the subchondral bone in a direction perpendicular to the joint surface. RESULTS: Graft step-off was statistically significantly better for the anatomic grafts compared to the generic grafts in the MFC (0.0 â€‹± â€‹0.2 â€‹mm vs. 0.7 â€‹± â€‹0.5 â€‹mm, p â€‹< â€‹0.001), LFC (0.1 â€‹± â€‹0.3 â€‹mm vs. 1.0 â€‹± â€‹0.2 â€‹mm, p â€‹< â€‹0.001), patella (-0.2 â€‹± â€‹0.3 â€‹mm vs. -1.2 â€‹± â€‹0.4 â€‹mm, p â€‹< â€‹0.001), and trochlea (-0.4 â€‹± â€‹0.3 vs. 0.4 â€‹± â€‹0.7, p â€‹= â€‹0.003). Graft contour was statistically significantly better for the anatomic grafts in the LFC (0.0 â€‹± â€‹0.0 â€‹mm vs. 0.2 â€‹± â€‹0.4 â€‹mm, p â€‹= â€‹0.022) and trochlea (0.0 â€‹± â€‹0.0 â€‹mm vs. 1.4 â€‹± â€‹0.7 â€‹mm, p â€‹< â€‹0.001). The anatomic grafts had an observed maximum step-off of -0.9 â€‹mm and a maximum contour mismatch of 0.8 â€‹mm. CONCLUSION: This study validates a process designed to fabricate anatomically accurate cartilage grafts using MRI and 3D printing technology. Anatomic grafts demonstrated superior fit compared to generic flat grafts. LEVEL OF EVIDENCE: Level IV.

4D Bioprinting via Molecular Network Contraction for Membranous Tissue Fabrication.

Generation of thin membranous tissues (TMT), such as the cornea, epidermis, and periosteum, presents a difficult fabrication challenge in tissue engineering (TE). TMTs consist of several cell layers that are less than 100 µm in thickness per layer. While traditional methods provide the necessary resolution for TMT fabrication, they require significant handling and incorporation of several layers is limited. Extrusion bioprinting offers precise control over deposition of different biomaterials and cell populations within the same construct but lacks the resolution to generate biomimetic TMTs. For the first time, a 4D bioprinting strategy that allows for the generation of cell-laden TMTs is developed. Anionic gelatin methacrylate (GelMA) hydrogels are treated with cationic poly-l-lysine (PLL), which induces charge attraction, microscale network collapse, and macroscale hydrogel shrinking. The impact of shrinking on hydrogel properties, print resolution, and cell viability is presented. Additionally, this work suggests that a novel mechanism is occurring, where PLL exhibits a contractile force on GelMA and PLL molecular weight drives GelMA shrinking capabilities. Finally, it is shown that this phenomenon can occur while maintaining an encapsulated cell population. These findings address a critical barrier by generating macroscale tissue structures with their microscale TMT counterparts in the same print.

Fabrication Strategies for Engineered Thin Membranous Tissues.

Thin membranous tissues (TMTs) are anatomical structures consisting of multiple stratified cell layers, each less than 100 µm in thickness. While these tissues are small in scale, they play critical roles in normal tissue function and healing. Examples of TMTs include the tympanic membrane, cornea, periosteum, and epidermis. Damage to these structures can be caused by trauma or congenital disabilities, resulting in hearing loss, blindness, dysfunctional bone development, and impaired wound repair, respectively. While autologous and allogeneic tissue sources for these membranes exist, they are significantly limited by availability and patient complications. Tissue engineering has therefore become a popular strategy for TMT replacement. However, due to their complex microscale architecture, TMTs are often difficult to replicate in a biomimetic manner. The critical challenge in TMT fabrication is balancing fine resolution with the ability to mimic complex target tissue anatomy. This Review reports existing TMT fabrication strategies, their resolution and material capabilities, cell and tissue response, and the advantages and disadvantages of each technique.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

An open-source bioink database for microextrusion 3D printing.

3D printing has rapidly become a critical enabling technology in tissue engineering and regenerative medicine for the fabrication of complex engineered tissues. 3D bioprinting, in particular, has advanced greatly to facilitate the incorporation of a broad spectrum of biomaterials along with cells and biomolecules of interest forin vitrotissue generation. The increasing complexity of novel bioink formulations and application-dependent printing conditions poses a significant challenge for replicating or innovating new bioprinting strategies. As the field continues to grow, it is imperative to establish a cohesive, open-source database that enables users to search through existing 3D printing formulations rapidly and efficiently. Through the efforts of the NIH/NIBIB Center for Engineering Complex Tissues, we have developed, to our knowledge, the first bioink database for extrusion-based 3D printing. The database is publicly available and allows users to search through and easily access information on biomaterials and cells specifically used in 3D printing. In order to enable a community-driven database growth, we have established an open-source portal for researchers to enter their publication information for addition into the database. Although the database has a broad range of capabilities, we demonstrate its utility by performing a comprehensive analysis of the printability domains of two well-established biomaterials in the printing world, namely poly(ϵ-caprolactone) and gelatin methacrylate. The database allowed us to rapidly identify combinations of extrusion pressure, temperature, and speed that have been used to print these biomaterials and more importantly, identify domains within which printing was not possible. The data also enabled correlation analysis between all the printing parameters, including needle size and type, that exhibited compatibility for cell-based 3D printing. Overall, this database is an extremely useful tool for the 3D printing and bioprinting community to advance their research and is an important step towards standardization in the field.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.

Dual Extrusion Patterning Drives Tissue Development Aesthetics and Shape Retention in 3D Printed Nipple-Areola Constructs.

Breast cancer and its most radical treatment, the mastectomy, significantly impose both physical transformations and emotional pain in thousands of women across the globe. Restoring the natural appearance of a nipple-areola complex directly on the reconstructed breast represents an important psychological healing experience for these women and remains an unresolved clinical challenge, as current restorative techniques render a flattened disfigured skin tab within a single year. To provide a long-term solution for nipple reconstruction, this work presents 3D printed hybrid scaffolds composed of complementary biodegradable gelatin methacrylate and synthetic non-degradable poly(ethylene) glycol hydrogels to foster the regeneration of a viable nipple-areola complex. In vitro results showcased the robust structural capacity and long-term shape retention of the nipple projection amidst internal fibroblastic contraction, while in vivo subcutaneous implantation of the 3D printed nipple-areola demonstrated minimal fibrotic encapsulation, neovascularization, and the formation of healthy granulation tissue. Envisioned as subdermal implants, these nipple-areola bioprinted regenerative grafts have the potential to transform the appearance of the newly reconstructed breast, reduce subsequent surgical intervention, and revolutionize breast reconstruction practices.

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.

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.

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.