Hands On Tissue Engineering Research
About the CI
Interested in working with cell and tissues for future clinical breakthroughs? This CI will provides hands-on experience. Students have worked on various projects involving the culture of novel three dimensional cancer models for screening purposes, creating cartilage tissues from chondrocyte cultures isolated from pig joints, development of a novel high throughput culture device base on the photoelectric effect of photovoltaic devices and more projects to come including cardiovascular tissues. This CI provides the insights of bioengineering to better assess the propensity to continue towards a more formal research environment at graduate school.
Current Projects
Because of the large premise of this creative inquiry, there are six individual projects that fall under the topic of tissue engineering. The current projects of this creative inquiry are listed below. Each tab holds more information on the focus of each project and where they are currently. Click on any project to see more!
3-D Brain Model
Creating a 3-D Model of the Brain
The aim of this project is to develop a patient-specific, time- and cost-effective, 3D brain model using materials that mimic the physical and mechanical properties of human brain tissue. This will aid surgeons in planning and predicting surgical outcomes with increased accuracy.
Personalized Cancer Medicine
High Throughput Screening With Modified Inkjet Bioprinter For Cancer Personalized Medicine
This project is working on the modification of a common inkjet printer. There are two sides to the outcome of this modification. The first is to successfully bio-print viable cancer cell cultures through the printer with a modified ink cartridge into well plates. The second aspect is to then run cytotoxic assays on those same cultures. High throughput screening is a method of evaluating a drug by rapidly assaying samples. Current high throughput screening machinery is very expensive, which limits its use to large pharmaceutical companies. The ultimate goal of this project is provide a novel method to rapidly produce cell cultures of a patient’s sample cells and then run cytotoxic assays on those same cultures with multiple different chemotherapeutic drugs. This will allow for a determination of most effective drug and dosage to that patient’s specific cell line. The method presented here will provide a rapid and cost effective alternative approach to personalized chemotherapeutic medicine and high throughput screening.
Engineering Vascular Grafts
Polymer Electrospinning
This project involves using a process known as electrospinning to create polymer fibers that will be molded to create a scaffold for a vascular graft. Electrospinning is a technique that uses electric charges to draw out condensed polymer solutions into fibers with diameters up to 10 nanometers. Once a high enough voltage h as been introduced to the solution, it becomes charged and surface tension is negated by electrostatic repulsion. This causes the droplet to be stretched until a critical point of liquid separates from the surface. This process is important for my project because electrospun fibers have been shown to be mechanically strong and biocompatible. The current focus is on different types of polymer combinations to determine which has the best ability to emulate living vascular mechanical properties. Spin casting, which involves using a motor to spin a collector in front of syringe excreting fibers, will be used to capture the fibers. The reasoning behind this is to align the fibers in a way that more closely resembles the alignment of vascular endothelium. Light microscopes can be used to observe differences in fiber alignment of samples. The project has also involved repurposing a robot used for dispensing gels for housing the collector and syringe for electrospinning. Once the scaffold for the graft is complete my plan is to implant living cells onto the graft so that they can survive and grow onto it.
Inhibiting Calcification of Vascular Grafts
When considering the electrospun fibers, we are also considering the best way to functionalize them in order to improve their utility. One common difficulty with vascular grafts is calcification, limiting the ability of the graft to mimic the pulsation nature of natural vasculature. This is especially the case when elastin is added to the graft prior to implantation. One method we are considering to diminish this effect is functionalizing polymers with poly-glutamylglutamine (PGG), which in literature has been shown to limit calcification. We are also considering AlCl3 for this purpose. In order to functionalize the polymers with PGG, we are first using an iron chloride assay to determine baseline levels of PGG absorbance, which will be used to determine the best concentration of PGG to functionalize the fibers with.
Cancer Microenvironment
Cancer Biomechanics
When cancer is biopsied, the cells remain cancerous outside of the body for only a finite period of time before they de-differentiate and lose the components that make them unique. This is due to the simple fact that cancer growth results from both cellular defects and the microenvironment of the tumor. When removed from the microenvironment the cells no longer behave as they do within the body, making treatment very difficult. In this Creative Inquiry, we aim to create a biomechanically sound cancer model outside of the body through the replication of the tumor microenvironment. If successful, a breakthrough such as this could revolutionize the field of cancer treatment, allowing for more tailored and targeted treatment of cancer from one individual to the next.
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Synthesizing the Dura Mater
Synthesizing the Dura Mater
This particular project deals with a creating a functional replica of the dura mater that can be used by medical students in a low-risk environment. The dura mater is the outermost membrane surrounding the brain. It lies between the brain and the skull and primarily serves to protect and nourish the brain. The synthesized dura mater itself is created using polymers and an electrospinning machine that weaves several layers together in order to make a replica that mimics the dura mater’s physical and mechanical properties. The creation of a synthetic alternative to the dura mater is not a new idea; however, this project’s aims, data, and production of the dura mater are all original.
In order to determine the properties of the dura mater, pig brains were used from obtained from Godley-Snell Research Center. Brains were harvested from the head of the pig at the time of death, and the dura mater was extracted from the skull. Then, the dura mater is cut from each side of the superior sagittal sinus. Next, the samples are loaded into a tensile test machine in order to test their tensile strength. The dura mater is a very thin, tough membrane, so the most important mechanical property it has is its ability to not tear so that it can protect the brain. This test gives the necessary data to create a substitute dura mater that has similar properties to the dura mater.
The next step in this project is to synthesize a suitable substitute to the dura mater. This is currently in development, but the plan is to electrospin several layers of porous membranes and then to weave them together. This allows the synthetic dura mater to have similar tensile properties as the real thing. Since this is still in development, suitable polymers have not yet been identified. However, due to the fact that there are several dura mater substitutes in the medical field, there are several polymers that have demonstrated favorable qualities for this project.
Cartilage Regeneration
Density and Mechanical Property Characterization of Chondrocyte Spheroids
Cartilage regeneration and implantation is a field of tissue engineering that is gaining large amounts of attention. The articular cartilage found in joints of the body lack the vascular components to bring growth factors to the sites of trauma. The current methods of repairing cartilage are joint lavage, abrasion arthroplasty, or the transplantation of autologous or allogeneic osteochondral grafts1, to name a few. However, through testing it seems that those approaches are not suitable to large defects or degenerative joint diseases like arthritis. In an effort to find another method, scientists have been working for some time on how to engineer tissue ex vivo made of chondroctyes, pluripotent stem cells, or mesenchymal stem cells. In addition, research has shown that 2D cell cultures are not physiologically relevant2 and “poorly mimic the conditions in the living organism.”3 Therefore other options have been tested, such as pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures. This research will focus on the use of chondrocyte spheroid cultures and their equivalency to articular cartilage.
An experiment carried out on the paracrine effect of transplanted rib chondrocyte spheroids showed that transplanted chondrocyte spheroids provide cells for repair tissue.4 Therefore it has been shown that chondrocyte spheroids can be used in certain applications for tissue regenerations. This project works to maximize the density of chondrocyte spheroids to simulate the chondrocytes in the native articular cartilage and develop those spheroids to have similar mechanical properties to that of the native articular cartilage. Pig tibiofemoral knee joints will be used to supply the chondrocytes because of the resemblance to human articular cartilage as well as
Currently the focus is on isolating chondrocytes from articular cartilage. The last effort to extract the chondrocytes failed–lack of attachment to the culturing flask–so the protocol is being adjusted to account for potential errors. Once the chondrocytes are being grown successfully in the lab, the next step is to find the spheroid density (cells/spheroid) and cell media combination that creates the most spherical chondrosphere. The densities that are going to be tested are as follows: 100,000; 500,000; and 750,000 cells/spheroid. The media that are going to be created include: DMEM with HEPES, Proline, ascorbic acid, fetal bovine serum, nonessential amino acids, antibiotic-antimycotic, and ampicillin B; and a second media that is supplemented with 0.24% methylcellulose.
Tibiofemoral joints after shaving of cartilage.
- Estes B, Diekman B, Gimble J, and Guilak F. Isolation of adipose derived stem cells and their induction to achondrogenic phenotype. Nat Protoc 5(7): 1294–1311.
- “2D Versus 3D Cell Culture.” 3D Biomatrix. 28 Sept 2015.
- Hess MW et al. 3D versus 2D cell culture implications for electron microscopy. Methods in Cell Biology. 2010, Elsevier.
Presentations and Awards
Cotton, B; McGreevey, J; Navarro, M; Advisors: Joseph, J; Dean, D; Rodriguez, JI. (2016) 3D Printed Brain Model for Practice and Preoperative Planning. GHS Poster Research Day. GHS memorial hospital
