Plan for Demonstrating Feasibility - Key Deliverables:

Personalized 3D Scaffold: The 3D printed scaffold using the reconstructed file generated from the optimization algorithm with the PCL-MSN solution will serve as our first main deliverable. Demonstrating the feasibility of the construct will start with obtaining publicly-available skull CT images that contain craniofacial defects. The initial algorithm will use the CT images as a test input, and then evaluate the accuracy of the computer-generated defects. Our team will subject the optimized scaffold to physical tests by creating a scaffold for a mock, medical-grade skull. The CT scan analysis will generate the solid, 3D outline of the specific defect., and SolidWorks will use the 3D outline as an input where the hollow interior will be replaced with repeated units of the ideal cross-sectional dimensions to maximize porosity without sacrificing mechanical strength. Upon examining the resulting design by overlaying the model into the original CT stack to ensure proper fit, the construct will be printed using the standard MakerBot. The Department of Mechanical Engineering and Applied Mechanics has available printers that like the ProJet 6000HD, which are able to print layer-by-layer with an accuracy of 0.025-0.05mm. As it is affordable and accessible, one of these high-resolution printers will be utilized to guarantee scaffold resolution of less than 1mm. Once printed, the scaffold will be tested for optimal release kinetics, mechanical properties, and overall fit inside the model skull.

Optimal Mechanical Properties: During the algorithm optimization phase, mechanical testing of the PCL-MSN polymer will be occurring simultaneously. The ideal deliverable through this testing will be that the scaffold itself is able to meet the minimal acceptable mechanical strength profiles of native bone and other clinically-approved scaffolds. First, Dynamic Mechanical Analysis (DMA) will be conducted with an Instron machine to generate a stress-strain curve in addition to modeling creep and stress-relaxation - all of which will provide information about the elastic modulus of the scaffold. The Instron machine will also be used to test the Ultimate Compressive Strength (UCS) of the material. We will test different compositions depending on PCL-MSN ratios and different levels of porosity to ensure the device is able to meet the desired mechanical thresholds. To measure these different levels of porosity, we will utilize a Scanning Electron Microscopy (SEM). This technique generates high resolution images by focusing electron particles at the outer surface of the scaffold. With these images, the size and percent porosity of the scaffold can be calculated and verified to better optimize the relationship between mechanical strength and porosity.

Chemical Testing: The first drug release profiles that will be observed are the antibiotics vancomycin and moxifloxacin, which are commonly used to treat bacterial infections in bones and joints. Because the scaffold are intended for clinical applications to treat cranial defects while overcoming the major drawbacks of current solutions such as high rates of infection, a significant overarching goal is to characterize antibody release profiles as accurately as possible. These drugs will be loaded within the xerogel of the construct for a slow-release profile over the course of 10 weeks to model the time period required for bone regeneration and scaffold degradation. Optimal loading profiles for these drugs will also be monitored to ensure the concentration never reaches an unsafe level as this could lead to negative side effects such as nephrotoxicity and ototoxicity. In-vitro studies using MSCs seeded on the 3D printed material will be conducted to ensure these proper release profiles by assessing cell viability, cell proliferation, and increased levels of bone regeneration as described in “Biological Testing”. Immunosuppressant drugs, such as cyclosporine A, will also be analyzed in a similar manner to limit initial immune response to the scaffold. Grafts and synthetic scaffolds often fail due to the high rates of rejection, so implanting the scaffold with these immunosuppressants will lead to greater mechanical strength of the scaffold and more sustained bone growth.

Biological Testing: MC3T3 cells are mouse osteoblast cells, which will be cultured onto the scaffold through two methods: (1) seeding after the PCL scaffold is printed and placed in a xerogel solution, or (2) direct seeding through bioprinting with cells in the same solution as the PCL:MSN, which will then be fed through the 3D printer. The latter mode of embedding cells has been explored by many labs, including that of Dr. Jason Burdick of the Department of Bioengineering. In order to ensure homogeneity, high retention rate, and direct integration within the macroenvironment, cells can be 3D printed in a cell culture hood. However, indirect seeding is beneficial in that cells naturally bind to PCL due to its intrinsic properties, and the viability would be higher than rough injection due to the large quantity of cells killed during printing. Cytotoxicity is a significant factor in determining if the 3D printed scaffolds are a viable alternative to the currently used materials - many metal scaffolds eventually fail. Experiments such as the LDH and MTS assays can be utilized to understand the viability of the cells and the cytotoxicity of the environment. For MTS assays, it is known that proliferating and viable cells reduce the concentration of MTS tetrazolium when exposed, so a high level of colorized MTS indicates high degrees of cytotoxicity. Therefore, these results are the most important in proving the feasibility of the project, as negative results would necessitate the reevaluation of every aspect of the scaffold to determine the limiting factor.

Current Progress:

Reconstruction: In terms of the reconstruction efforts so far, we have been able to make some major improvements that will allow for less required work next semester. First, we were able to access publicly-available CT images online and download several sets of .dicom images of healthy skulls. With this data, we have been able to successfully read-in 3D CT and separate images on a slice-by-slice basis for reconstruction. Within these individual slices, we have been able to make significant progress in our reconstruction efforts. As seen in the image, the progress to this point can be visualized in six steps. (1) shows our ability to read in CT data on a slice-by-slice basis for reconstruction. Step (2) shows a conversion of the CT image to binary, using the most sensitive possible threshold, which was advised by Dr. Ari Brooks. Image panes (3) and (4) show our capabilities to create outlines of the skull depending on different borders and sides of the skull, which will be helpful when localizing defects in 2D. (5) represents current geometrical modelling that we have employed by using code to fit an ellipse to the inner and outer borders of the skull through a least-squares regression. Finally, (6) shows that, with these geometrical models, we can determine certain properties of the skull, such as the labeled axis of rotation.

Biologics: Biologically, we accomplished one of the important principle synthesis reactions necessary for our scaffold’s manufactured drug release profile: fabrication of blank mesoporous silica nanoparticles. We mimicked a method developed by Sanjib Bhattacharyya, Henson Wang, and Paul Ducheyne as detailed in “Polymer-Coated Mesoporous Silica Nanoparticles for the Controlled Release of Macromolecules” in Acta Biomaterialia. Citation/footnote (I have paper). First, C₁₈TAB (octadecyltrimethylammoniumbromide) was dissolved in distilled water at 75℃, and then mixed with 2M NaOH (sodium hydroxide) to make a basic solution with pH 12. The main molecule, TEOS (tetraethylorthosilicate) was dissolved in with drops of TESPA (3-(triethoxlysilyl) propyl-succinic anhydride), and the solution was left to dry for two days. The dried MSN particles were added to aqueous DMH (dimethylhexadecylamine), stirred, and then heated. The leftover solid was rinsed with methanol first, followed by rinses with ethanol and water. This procedure creates pore-expanded, unloaded MSN particles which can take on a biologic to load its pores  when we design the PCL/MSN solution. The figure Number(MSN stages graphic, obtained from paper) above shows the main stages of the MSN fabrication and loading process with the last step of the PEG-coated MSN as the final product necessary to produce the manufactured drug release profile.