1. Hydrogel-based in-vitro spheroid models of glioblastoma: towards development of predictive drug screening platforms

 

 

       Glioblastoma multiforme (GBM) is a lethal disease with poor patient prognosis. GBM is the most aggressive type of brain tumor, with median patient survival of only 12-15 months. Over 23,000 new cases of central nervous system tumors are reported, and over 14,000 deaths occur annually due to GBM in the United States. Typical treatments include maximum safe surgical resection of bulk tumor and chemotherapy with temozolomide (TMZ), coupled with radiation therapy. TMZ is currently the only approved GBM chemotherapy, and provides only a modest two month increase in median survival. Anti-angiogenesis therapy via bevacizumab showed promising results at early clinical trials, but phase III trials revealed increased toxic side effects without additional overall survival. These circumstances demonstrate the lack of effective GBM therapy and warrant further research. To facilitate such research, in vitro bioengineered brain models that adequately recapitulate aspects of the in vivo tumor microenvironment are greatly needed. The goal of this project is to develop a novel hydrogel-based GBM spheroid model, as well as to characterize spheroid outcomes including: i) cancer stem cell enrichment, ii) extracellular matrix secretion by the cancer cells, and iii) drug responsiveness. These outcomes are characterized as a function of tunable hydrogel properties. By establishing key matrix properties that affect the above outcomes, we are filling a toolbox for better in vitro drug screening.

 

Collaborators: Dr. Yonghyun Kim, Chemical and Biological Engineering, University of Alabama

                       Prof. Yancey Gillespie, Neurosurgery, University of Alabama at Birmingham

 

             2. Development of carbon nanotube-hydrogel nanocomposites as scaffolds for neural tissue engineering

 

 

             Neural injuries are devastating and mostly incurable diseases affecting millions of Americans and costing billions of dollars in medical expenditures. Interestingly, while mammalian neurons possess an intrinsic regenerative capacity, most patients with neural injuries never regain function due to the inability of the healthy part of the neuron to cross the injury site and re-connect with its distal counterpart. Much research has, thus, focused on developing permissive biomaterial substrates that can guide and support neuronal regeneration and growth through the injury site, specifically focusing on the macro and micro properties of the biomaterial scaffolds. However, recent discoveries have demonstrated that to fully harvest the biomaterial potential, a scaffold must replicate all length scales of the cellular environment, including the nanoscale. With this in mind, we are developing novel carbon nanotube-hydrogel nanocomposites which are capable of supporting cell attachment and spreading even in the absence of any natural cell adhesive ligands.    A novel method to transfer of single wall carbon nanotubes onto hydrogels: For the very first time, we were able to transfer a dense meshwork of single wall high quality, semi-conducting carbon nanotubes on soft hydrogel scaffolds that have the bulk stiffness of brain tissue. The carbon nanotubes themselves are several microns long but only 1-2 nm in diameter. For comparison, one of the most common adhesive ligands (a short amino acid sequence present in several fibrous extracellular matrix proteins) that cells can recognize and bind to is only 1 nm in diameter. Hence, we believe that introducing such nano length scale in a bulk scaffold of physiologically-relevant properties will enable us to create an optimum cellular environment that will enable unprecedented neural growth and regeneration.

             Carbon nanotube hydrogel composites as electroconductive 3D cell scaffolds: In another related project, we are developing carbon nanotube-polyethylene glycol composites as a scaffold for neural cell encapsulation. Specifically, we are devising methods to achieve even dispersion and alignment of carbon nanotubes into the hydrogels for 3D cell scaffolds. Further, the hydrogel ability to support neural cell adhesion and neurite extension is being addressed. The ultimate goal of the project is to build a semi-conductive platform for directed neuronal growth and electrical stimulation of neural cells that more closely mimics the in vivo cell environment and thus could serve as a viable in vitro model for the study of neural tissue physiology and pathology.

 

Collaborators: Dr. Irma Kuljanishvili, Physics, St Louis University

                       Dr. Fenglian Xu, Biology, St Louis University

 

       3. Decoupling key cell-matrix interactions that affect cell responsiveness to therapeutics in a bioengineered extracellular matrix

 

The goal of this project is to test the synergistic or antagonistic effects of matrix parameters such as dimensionality, stiffness, adhesive ligand presentation and drug penetration limitations on cell responses to drugs. The results will provide a toolbox for the rational design of predictive drug screening platforms. Current research suggests that encapsulating cells in biomaterial matrices alters the cellular response to drugs; however, it is unclear whether there are minimum requirements (e.g., physiologically relevant dimensionality and stiffness) that can significantly improve predictive capacity of biomaterial-based drug screening platforms and whether these requirements are universal or specific to disease and drug type combinations. Our aim is to comprehensively investigate: 1) the role of a biomaterial matrix and its properties on cell drug responsiveness,  and 2) cell-secreted ECM as a function of matrix properties and its correlation to hindered drug diffusivity and cell drug responses. Matrices with physiologic mechanical and biochemical properties, even though not fully recapitulating the in vivo environment, could improve drug screening efficiency by narrowing the list of candidates for in vivo validation, reducing time and cost of drug development.

Collaborator: Dr. Joseph Schober, Pharmaceutical Sciences, Southern Illinois University, Edwardsville

 

              4. Injectable hydrogel microspheres for sustained protein delivery

 

     While our main focus is on the technology development, we have two application-based collaborative projects.

             PRP delivery for the treatment of knee osteoarthritis: Knee osteoarthritis (KOA) is an increasingly prevalent condition with a significant negative impact on quality of life via debilitating joint pain caused by articular cartilage degeneration. Available clinical treatments are mostly palliative and fail to suppress cartilage degeneration or promote tissue regeneration. Platelet-rich plasma (PRP) therapy has shown promise in accelerating tissue remodeling in vitro but has received mixed results in the clinic due to inefficient delivery. Bolus delivery is inadequate due to rapid clearance and attempted sustained delivery systems, such as gelatin and alginate microspheres, offer limited tunability of PRP release kinetics for optimal impact. The aim of this project is to fabricate a novel injectable drug delivery system based on tunable polyethylene glycol microspheres for temporally relevant delivery of PRP biomolecules to the osteoarthritic. This novel delivery system may have broader impact in the treatment of a range of arthritic conditions.

             GALNS Enzyme Replacement Therapy: We also employ our injectable microgels for the encapsulation and delivery of N-acetylgalactosamine 6-sulfatase (GALNS) enzyme with the broader goal of developing an effective enzyme replacement therapy. Deficiency of GALNS causes accumulation of glycosoaminoglycans and ultimately Morquio A disease. Although effective in alleviating some disease symptoms, current enzyme replacement therapy has many limitations, such as a need for recurrent infusions and occurrence of immune responses. Delivery of GALNS encapsulated in microgels could offer an improvement over existing therapies, as it preserves enzyme activity, leads to longer circulation times and attenuates immune responses, while requiring a single injection.

 

Collaborators: Dr. Adriana Montaño, Pediatrics, St Louis University Medical School

                       Dr. Scott Sell, Biomedical Engineering, St Louis University

                       Dr. Muhammad Farooq Rai, Orthopedic Surgery, Washington University in St Louis

                       Dr. Linda Sandell, Orthopedic Surgery, Washington University in St Louis

     

              5. Transport in complex media: real time measurements and new predictive models

    

            

             Fluorescence Correlation Spectroscopy (FCS) for the study of diffusion in complex media: Diffusion of oxygen, nutrients and other biomolecules is critical for cell survival and is one of the major obstacles in building 3D tissue engineering scaffolds. However, due to technical challenges, few techniques are capable of performing diffusion measurements in such complex 3D environments. In our lab, we adapt and use FCS. FCS measures fluctuations in the fluorescence intensity of solutes moving through a small illuminated sample volume. The fluctuations are correlated in time by a time-delayed autocorrelation function, which is a measure of the concentration and translational diffusivity of the fluorescent solute. The advantages of FCS are numerous: non-invasive measurements of nanomolar solute concentrations, direct estimation of solute concentration, scaffold structure, scaffold dynamics such as swelling behavior, solute-solute, and solute-matrix interactions.

             Prediction of macroscale solute movement from finescale structure in aqueous polymer environments: This project aims to develop techniques for prediction of macroscale diffusivity of a dilute solute molecule from the description of finescale structure of the polymer environment. We aim to develop finescale models of various categories of environments and of varying degrees of detail and then systematically investigate computational and theoretical approaches based on Monte Carlo simulations as well as homogenization theory to obtain approximate macroscale solute  diffusivity. The goal of this study is to provide a clear and rigorous understanding of how finescale polymer system properties affect macroscale solute movement.

 

Collaborator: Prof. Muruhan Rathinam, Mathematics, University of Maryland Baltimore County

 

             6. Hydrogel Biomaterials for Islet Encapsulation and Implantation

 

Approximately 1.25 million American children and adults have type 1 diabetes mellitus (T1DM), which is a chronic, progressive autoimmune disease caused by selective destruction of insulin-producing -cells within the pancreatic islets of Langerhans. Insulin delivered through an insulin pump or daily injections is essential to sustain life for these patients. Despite careful monitoring of blood glucose levels, a subset of patients with complicated T1DM have high risks of experiencing life threatening hypoglycemia episodes. One emerging new treatment for these patients include -cell replacement therapy. Islet transplantation, when successful, eliminates hypoglycemic episodes, slows or prevents the progression of complications, and improves quality of life for patients. However, major hurdles for this therapy include poor islet survival, side effects of life-long immunosuppressant administration, and shortage of donor islets. To circumvent islet shortage and rejection post transplantation, islet encapsulation within semi-permeable, biocompatible membrane as a strategy to hide islets from host immune cells has emerged. However, there is still a gap to meet challenges of prolonging islet survival due to lack of adequate oxygen and nutrients supply and a lack of protection from small diffusible molecules such as cytotoxic cytokines and pro-inflammatory mediators. Our research aims to address the challenge of prolonging encapsulated islet survival post-implantation using strategies of first creating a local vascularized islet implantation site, followed by injection of islets into the pre-vascularized site. This strategy is anticipated to promote revascularization within islets post transplantation, supplying adequate oxygen and nutrients, thus, enhancing islet engraftment and survival.

 

Collaborator: Dr. Guim Kwon, Pharmaceutical Sciences, Southern Illinois University of Edwardsville

 

 

 

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Dr. Silviya Petrova Zustiak, Associate Professor, Biomedical Engineering, Saint Louis University.

3507 Lindell Blvd, St. Louis, MO 63103-2010. Phone: (314) 977-8331. E-mail: silviya.zustiak@slu.edu

Zustiak Soft Tissue Engineering Laboratory

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