A 3D-printed blood-brain barrier model with tunable topology and cell-matrix interactions.

Recent developments in digital light processing (DLP) can advance the structural and biochemical complexity of perfusablein vitromodels of the blood-brain barrier. Here, we describe a strategy to functionalize complex, DLP-printed vascular models with multiple peptide motifs in a single hydrogel. Different peptides can be clicked into the walls of distinct topologies, or the peptide motifs lining channel walls can differ from those in the bulk of the hydrogel. The flexibility of this approach is used to both characterize the effects of various bioactive domains on endothelial coverage and tight junction formation, in addition to facilitating astrocyte attachment in the hydrogel surrounding the endothelialized vessel to mimic endothelial-astrocyte interaction. Peptides derived from proteins mediating cell-extracellular matrix (e.g. RGD and IKVAV) and cell-cell (e.g. HAVDI) adhesions are used to mediate endothelial cell attachment and coverage. HAVDI and IKVAV-lined channels exhibit significantly greater endothelialization and increased zonula-occluden-1 (ZO-1) localization to cell-cell junctions of endothelial cells, indicative of tight junction formation. RGD is then used in the bulk hydrogel to create an endothelial-astrocyte co-culture model of the blood-brain barrier that overcomes the limitations of previous platforms incapable of complex topology or tunable bioactive domains. This approach yields an adjustable, biofabricated platform to interrogate the effects of cell-matrix interaction on blood-brain barrier mechanobiology.

Porous Scaffold-Hydrogel Composites Spatially Regulate 3D Cellular Mechanosensing.

Cells encapsulated in 3D hydrogels exhibit differences in cellular mechanosensing based on their ability to remodel their surrounding hydrogel environment. Although cells in tissue interfaces feature a range of mechanosensitive states, it is challenging to recreate this in 3D biomaterials. Human mesenchymal stem cells (MSCs) encapsulated in methacrylated gelatin (GelMe) hydrogels remodel their local hydrogel environment in a time-dependent manner, with a significant increase in cell volume and nuclear Yes-associated protein (YAP) localization between 3 and 5 days in culture. A finite element analysis model of compression showed spatial differences in hydrogel stress of compressed GelMe hydrogels, and MSC-laden GelMe hydrogels were compressed (0-50%) for 3 days to evaluate the role of spatial differences in hydrogel stress on 3D cellular mechanosensing. MSCs in the edge (high stress) were significantly larger, less round, and had increased nuclear YAP in comparison to MSCs in the center (low stress) of 25% compressed GelMe hydrogels. At 50% compression, GelMe hydrogels were under high stress throughout, and this resulted in a consistent increase in MSC volume and nuclear YAP across the entire hydrogel. To recreate heterogeneous mechanical signals present in tissue interfaces, porous polycaprolactone (PCL) scaffolds were perfused with an MSC-laden GelMe hydrogel solution. MSCs in different pore diameter (~280-430 µm) constructs showed an increased range in morphology and nuclear YAP with increasing pore size. Hydrogel stress influences MSC mechanosensing, and porous scaffold-hydrogel composites that expose MSCs to diverse mechanical signals are a unique biomaterial for studying and designing tissue interfaces.

Effect of temperature and ultraviolet light on the bacterial kill effectiveness of antibiotic-infused 3D printed implants.

Drug eluting 3D printed polymeric implants have great potential in orthopaedic applications since they are relatively inexpensive and can be designed to be patient specific thereby providing quality care. Fused Deposition Modeling (FDM) and Stereolithography (SLA) are among the most popular techniques available to print such polymeric implants. These techniques facilitate introducing antibiotics into the material at microscales during the manufacturing stage and subsequently, the printed implants can be engineered to release drugs in a controlled manner. However, FDM uses high temperature to melt the filament as it passes through the nozzle and SLA relies on exposure to nanoscale wavelength ultraviolet (UV) light which can adversely affect the anti-bacterial effectiveness of the antibiotics. The focus of this article is two-fold: i) Examine the effect of high temperature on the bacterial kill-effectiveness of eluted antibiotics through Polycaprolactone (PCL) based femoral implants and ii) Examine the effect of exposure to ultraviolet (UV) light on the bacterial kill-effectiveness of eluted antibiotics through femoral implants made up of a composite resin with various weight fractions of Polyethylene Glycol (PEG) and Polyethylene Glycol Diacrylate (PEGDA). Results indicate that even after exposing doxycycline, vancomycin and cefazolin at different temperatures between 20oC and 230oC, the antibiotics did not lose their effectiveness (kill radius of at least 0.85 cm). For doxycycline infused implants exposed to UV light, it was seen that a resin with 20 % PEGDA and 80 % PEG had the highest efficacy (1.8 cm of kill radius) and the lowest efficacy was found in an implant with 100 % PEGDA (1.2 cm of kill radius).

Efficacy of eluted antibiotics through 3D printed femoral implants.

Costs associated with musculoskeletal diseases in the United States account for 5.7% of the Gross Domestic Product (GDP) (Weinstein et al. 2018). As such, there is a need to pursue new ideas in orthopaedic implants that can decrease cost and improve patient care. In the recent years, 3D printing of polymers using Fused Deposition Modeling (FDM) and metals using Direct Metal Laser Sintering (DMLS) has opened several exciting possibilities to create customized orthopaedic implants. Such implants can be engineered to release antibiotics in a controlled manner by infusing the drug into the material during manufacturing stage. However, the prevalence of high temperature could impact the anti-bacterial effectiveness of the eluted antibiotics in such implants. An alternative approach to circumvent this issue would be to modify the implant geometry to incorporate built-in design features such as micro-channels and reservoirs in which antibiotics can be introduced prior to the surgical procedure. Irrespective of the approach used, the ability of 3D printed orthopaedic implants to elute antibiotics, and the rate of elution are not well understood. The purpose of this article is to study the elution of doxycycline through 3D printed femoral implants using three different materials: Poly-Lactic Acid (PLA), Poly-Caprolactone (PCL) and Titanium grade Ti-6Al-4V. The PLA and Ti-6Al-4V implants were designed with built-in reservoirs and micro-channels in which doxycycline was introduced post the manufacturing stage. However, the PCL implants were printed from a PCL spool that was infused with doxycycline using an extruder. The PLA and Ti-6Al-4V experiments were run for a period of 31 days and the PCL experiment for one day. The antibacterial ability of eluted doxycycline from all implants were examined using Kirby-Bauer test on the bacteria E.coli k-12. The results show that most of doxycycline eluted through the three materials in the first 24 hours. After the initial spike, a steady release was achieved for the PLA and Ti-6Al-4V implants for 30 days. During this timeframe, Ti-6Al-4V implants released more doxycycline than the PLA implant. The eluted antibiotics through all the implants demonstrated the ability to kill bacteria in the subsequent Kirby-Bauer test. These outcomes show that irrespective of how the antibiotics were introduced, 3D printed polymeric and metallic implants have great potential in orthopaedic applications.