Fabricating vascularized, anatomically accurate bone grafts using 3D bioprinted sectional bone modules, in-situ angiogenesis, BMP-2 controlled release, and bioassembly.

Bone grafting is the most common treatment for repairing bone defects. However, current bone grafting methods have several drawbacks. Bone tissue engineering emerges as a promising solution to these problems. An ideal engineered bone graft should exhibit high mechanical strength, osteogenic properties, and pre-vascularization. Both top-down (using bulk scaffold) and bottom-up (using granular modules) approaches face challenges in fulfilling these requirements. In this paper, we propose a novel sectional modular bone approach to construct osteogenic, pre-vascularized bone grafts in anatomical shapes. We 3D-printed a series of rigid, thin, sectional, porous scaffolds from a biodegradable polymer, tailored to the dimensions of a femur bone shaft. These thin sectional modules promote efficient nutrition and waste removal due to a shorter diffusion distance. The modules were pre-vascularized viain-situangiogenesis, achieved through endothelial cell sprouting from the scaffold struts. Angiogenesis was further enhanced through co-culture with bioprinted fibroblast microtissues, which secreted pre-angiogenic growth factors. Sectional modules were assembled around a porous rod incorporated with Bone Morphogenetic Protein-2 (BMP-2), which released over 3 weeks, demonstrating sustained osteogenic activity. The assembled scaffold, in the anatomical shape of a human femur shaft, was pre-vascularized, osteogenic, and possessed high mechanical strength, supporting 12 times the average body weight. The feasibility of implanting the assembled bone graft was demonstrated using a 3D-printed femur bone defect model. Our method provides a novel modular engineering approach for regenerating tissues that require high mechanical strength and vascularization.

Cellular Patterning Alone Using Bioprinting Regenerates Articular Cartilage Through Native-Like Cartilagenesis.

Few studies have proved that bioprinting itself helps recapitulate native tissue functions mainly because the bioprinted macro shape can rarely, if ever, influence cell function. This can be more problematic in bioprinting cartilage, generally considered more challenging to engineer. Here a new method is shown to micro-pattern chondrocytes within bioprinted sub-millimeter micro tissues, denoted as patterned micro-articular-cartilages tissues (PA-MCTs). Under the sole influence of bioprinted cellular patterns. A pattern scoring system is developed after over 600 bioprinted cellular patterns are analyzed. The top-scored pattern mimics that of the isogenous group in native articular cartilage. Under the sole influence of this pattern during PA-MCTs bio-assembling into macro-cartilage and repairing cartilage defects, chondrogenic cell phenotype is preserved, and cartilagenesis is initiated and maintained. Neocartilage tissues from individual and assembled PA-MCTs are comparable to native articular cartilage and superior to cartilage bioprinted with homogeneously distributed cells in morphology, biochemical components, cartilage-specific protein and gene expression, mechanical properties, integration with host tissues, zonation forming and stem cell chondrogenesis. PA-MCTs can also be used as osteoarthritic and healthy cartilage models for therapeutic drug screening and cartilage development studies. This cellular patterning technique can pave a new way for bioprinting to recapitulate native tissue functions via tissue genesis.

Articular Cartilage Regeneration through Bioassembling Spherical Micro-Cartilage Building Blocks.

Articular cartilage lesions are prevalent and affect one out of seven American adults and many young patients. Cartilage is not capable of regeneration on its own. Existing therapeutic approaches for articular cartilage lesions have limitations. Cartilage tissue engineering is a promising approach for regenerating articular neocartilage. Bioassembly is an emerging technology that uses microtissues or micro-precursor tissues as building blocks to construct a macro-tissue. We summarize and highlight the application of bioassembly technology in regenerating articular cartilage. We discuss the advantages of bioassembly and present two types of building blocks: multiple cellular scaffold-free spheroids and cell-laden polymer or hydrogel microspheres. We present techniques for generating building blocks and bioassembly methods, including bioprinting and non-bioprinting techniques. Using a data set of 5069 articles from the last 28 years of literature, we analyzed seven categories of related research, and the year trends are presented. The limitations and future directions of this technology are also discussed.

3D-printed insert-array and 3D-coculture-array for high-throughput screening of cell migration and application to study molecular and cellular influences.

Collective cell migration refers to the movement of groups of cells and collective cell behavior and relies on cell-cell communication and cell-environment interactions. Collective cell migration plays a fundamental role in many aspects of cell biology and pathology. Current protocols for studying collective cell migration either use destructive methods or are not convenient for liquid handling. Here we present a novel 3D-printed insert-array and a 3D-coculture-array for collective cell migration study in high-throughput. The fabricated insert-array is comprised of 96 cylinder shaped inserts which can be placed in each well of a 96-well plate generating watertight contact with the bottom of each well. The insert-array has high manufacturing tolerance, and the coefficient of variations of the insert diameter and circularity are 0.67% and 0.03%, respectively. Each insert generates a circular cell-free area within the well without cell damage and provides convenient access for both manual and robotic liquid handling. Using the 3D-printed insert-array, we studied the migration of human umbilical vein endothelial cells (HUVECs) under the molecular influences of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and under the cellular influences of human mesenchymal stem cells (hMSCs) using the 3D-coculture-array. Our results show that the migration of HUVECs was dose-dependent on the VEGF and bFGF with different correlation patterns. They also generated a synergic pro-migration effect. When cocultured with hMSCs, the migration rate increased significantly while dependent on the number of hMSCs. The effects were partially blocked by VEGF inhibitor which suggests that VEGF secreted from hMSCs plays an important role in cell-to-cell communication during cell migration. The 3D-coculture-array can be manufactured at very low cost and shows higher biomolecule transport efficiency than the commercially available transwell. The calculated Z-factor is 0.66, which classifies our system as a perfect high-throughput assay. In summary, our newly developed insert-array and 3D-coculture-array provide a versatile platform to study collective cell migration in high-throughput as well as the molecular and cellular influences upon it.

A Novel 3D Bioprinter Using Direct-Volumetric Drop-On-Demand Technology for Fabricating Micro-Tissues and Drug-Delivery.

Drop-on-demand (DOD) 3D bioprinting technologies currently hold the greatest promise for generating functional tissues for clinical use and for drug development. However, existing DOD 3D bioprinting technologies have three main limitations: (1) droplet volume inconsistency; (2) the ability to print only bioinks with low cell concentrations and low viscosity; and (3) problems with cell viability when dispensed under high pressure. We report our success developing a novel direct-volumetric DOD (DVDOD) 3D bioprinting technology that overcomes each of these limitations. DVDOD can produce droplets of bioink from < 10 nL in volume using a direct-volumetric mechanism with < ± 5% volumetric percent accuracy in an accurate spatially controlled manner. DVDOD has the capability of dispensing bioinks with high concentrations of cells and/or high viscosity biomaterials in either low- or high-throughput modes. The cells are subjected to a low pressure during the bioprinting process for a very short period of time that does not negatively impact cell viability. We demonstrated the functions of the bioprinter in two distinct manners: (1) by using a high-throughput drug-delivery model; and (2) by bioprinting micro-tissues using a variety of different cell types, including functional micro-tissues of bone, cancer, and induced pluripotent stem cells. Our DVDOD technology demonstrates a promising platform for generating many types of tissues and drug-delivery models.

Rapid Fabrication of Anatomically-Shaped Bone Scaffolds Using Indirect 3D Printing and Perfusion Techniques.

Fused deposit modeling (FDM) 3D printing technology cannot generate scaffolds with high porosity while maintaining good integrity, anatomical-surface detail, or high surface area-to-volume ratio (S/V). Solvent casting and particulate leaching (SCPL) technique generates scaffolds with high porosity and high S/V. However, it is challenging to generate complex-shaped scaffolds; and solvent, particle and residual water removal are time consuming. Here we report techniques surmounting these problems, successfully generating a highly porous scaffold with the anatomical-shape characteristics of a human femur by polylactic acid polymer (PLA) and PLA-hydroxyapatite (HA) casting and salt leaching. The mold is water soluble and is easily removable. By perfusing with ethanol, water, and dry air sequentially, the solvent, salt, and residual water were removed 20 fold faster than utilizing conventional methods. The porosities are uniform throughout the femoral shaped scaffold generated with PLA or PLA-HA. Both scaffolds demonstrated good biocompatibility with the pre-osteoblasts (MC3T3-E1) fully attaching to the scaffold within 8 h. The cells demonstrated high viability and proliferation throughout the entire time course. The HA-incorporated scaffolds demonstrated significantly higher compressive strength, modulus and osteoinductivity as evidenced by higher levels of alkaline-phosphatase activity and calcium deposition. When 3D printing a 3D model at 95% porosity or above, our technology preserves integrity and surface detail when compared with FDM-generated scaffolds. Our technology can also generate scaffolds with a 31 fold larger S/V than FDM. We have developed a technology that is a versatile tool in creating personalized, patient-specific bone graft scaffolds efficiently with high porosity, good scaffold integrity, high anatomical-shaped surface detail and large S/V.

Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening.

Collective cell migration, in which cells migrate as a group, is fundamental in many biological and pathological processes. There is increasing interest in studying the collective cell migration in high throughput. Cell scratching, insertion blocker, and gel-dissolving techniques are some methodologies used previously. However, these methods have the drawbacks of cell damage, substrate surface alteration, limitation in medium exchange, and solvent interference. The superhydrophobic surface, on which the water contact angle is greater than 150 degrees, has been recently utilized to generate patterned arrays. Independent cell culture areas can be generated on a substrate that functions the same as a conventional multiple well plate. However, so far there has been no report on superhydrophobic patterning for the study of cell migration. In this study, we report on the successful development of a robotically patterned superhydrophobic array for studying collective cell migration in high throughput. The array was developed on a rectangular single-well cell culture plate consisting of hydrophilic flat microwells separated by the superhydrophobic surface. The manufacturing process is robotic and includes patterning discrete protective masks to the substrate using 3D printing, robotic spray coating of silica nanoparticles, robotic mask removal, robotic mini silicone blocker patterning, automatic cell seeding, and liquid handling. Compared with a standard 96-well plate, our system increases the throughput by 2.25-fold and generates a cell-free area in each well non-destructively. Our system also demonstrates higher efficiency than conventional way of liquid handling using microwell plates, and shorter processing time than manual operating in migration assays. The superhydrophobic surface had no negative impact on cell viability. Using our system, we studied the collective migration of human umbilical vein endothelial cells and cancer cells using assays of endpoint quantification, dynamic cell tracking, and migration quantification following varied drug treatments. This system provides a versatile platform to study collective cell migration in high throughput for a broad range of applications.