Regenerative Medicine

 

Cell-Extracellular Matrix Interactions

 

The extracellular matrix (ECM) contains an abundant variety of signals that are received by cell surface receptors and contribute to cell adhesion and cell fate, via regulation of cellular activities such as proliferation, migration and differentiation. As such, regenerative medicine studies often rely on mimicking the natural ECM to promote the formation of new tissue by host cells, and characterization of natural ECM components is vital for the development of new biomimetic approaches. A wide array of bioactive molecules contribute to cell-ECM interactions, including integrins, glycosaminoglycans, glycoproteins, fiber-forming elements, elastins and collagens. In our studies, we utilize synthetic peptide nanofiber systems to mimic the function of these matrix components and try to investigate extracellular matrix-cell interactions by using biofunctional nanofibers. Our group functionalizes peptide scaffolds with ligands of cell surface receptors and analyzes the effects of these interactions at a molecular level. We have also designed and synthesized peptide nanofiber systems with various functionalities, such as glycosaminoglycan mimics with growth factor binding capability, and shown that these nanofiber systems, together with other functional nanofibers, contribute to the growth and differentiation during neural differentiation, chondrogenesis and angiogenesis.

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Biomaterials for Regenerative Medicine

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Phage display is a technique that investigates the interactions between proteins, peptides and nucleic acids by engineering the natural properties of bacteriophages. Phages are bacterium-infecting viruses that direct the synthesis of proteins based on their own genetic information. This property allows the production of a specific protein or peptide on the coat protein of a genetically modified phage. The protein/peptide of interest can then be screened against a target molecule for the detection of specific interactions between biological molecules. Commercially available libraries, which contain large numbers of peptide molecules to expressed on virus surfaces, are commonly employed to find sequences that bind to a target of interest with high specificity. Our previous studies have demonstrated the importance of peptide amphiphile-growth factor interactions for the regeneration of several tissue types, such as nerve, cartilage, bone and skin. We are now employing phage libraries to better manipulate cell-peptide material interactions and further enhance the regenerative efficiency of our peptide amphiphile networks.

 

Stem Cell Biology

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Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of differentiating into a wide variety of distinctive end-stage cell types, including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes, endothelial cells and neuronal cells. In adults, their multipotency is of considerable importance for the regeneration of damaged or diseased mesenchymal tissues. The ability of MSCs to differentiate into a vast array of cell types, as well as their ease of manipulation in cell culture conditions, make them ideal candidates for the development of several therapeutic applications. Therefore, a thorough investigation of the regenerative potential of mesenchymal stem cells is necessary for the future of regenerative medicine and tissue engineering. Our laboratory focuses on the development of novel regenerative therapies by combining bioengineering strategies and stem cell biology, and aims to direct MSCs to specific lineages by utilizing 2- or 3-dimensional biomimetic microenvironments that share similar physical properties and bioactive ligands with native extracellular matrices. We have already designed peptide amphiphiles capable of triggering differentiation into osseous and cartilaginous tissues, and are currently working on the further development of extracellular matrix mimetic functional scaffolds, as well as the characterization of their impacts on the differentiation of MSCs.

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Neural Tissue Regeneration

 

As the nervous system is responsible for the regulation and coordination of all other bodily systems, symptoms associated with neural tissue damage tend to be exceptionally severe. Recent insights into the physiology of the nervous system suggest that neuronal regeneration may occur much more frequently than previously thought, but many nervous system diseases and injuries remain untreatable by current medical procedures. Following the establishment of the field of regenerative medicine, biomimetic molecules have received much attention as potential effectors of neural regeneration, and short peptide signals present in the neural extracellular matrix (ECM) are particularly promising in this context. Our study area is the development of ECM-mimetic peptide nanofiber scaffolds for the enhancement of neural differentiation and proliferation. To this end, we synthesize a variety of scaffolds that incorporate bioactive peptide sequences derived from neural ECM components, with emphasis on laminin-derived peptide nanofiber networks. These nanofibers, when used in conjunction with heparan sulfate-mimetic sulfonated peptides, were shown to stimulate the production of neurite outgrowths in PC-12 cells.

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Biomineralization

 

Biomineralization, the deposition of minerals by cells into their extracellular matrix (ECM), constitutes the essential mechanism of bone and teeth formation in humans and is closely involved in the regeneration of bone defects. Both collagenous and non-collageneous ECM molecules regulate cellular behavior, such as adhesion, migration, proliferation and differentiation, during the biomineralization process. Glycosaminoglycans (GAGs), one of the non-collageneous components of the ECM, play a particularly important role in bone remodeling and are responsible for stabilizing growth factors and enhancing growth factor-receptor interactions, thereby affecting cellular proliferation and differentiation. In our studies, peptide amphiphile molecules were designed and synthesized in order to mimic the presence of specific GAGs and collagen types in the ECM, thus encouraging the regeneration of osseous tissues in damage sites. A nanofibrous hydrogel scaffold was formed by mixing negatively and positively charged peptides, and demonstrated to serve as an effective regulator of cellular processes such as angiogenesis and osteogenic differentiation. We are currently investigating the effects of GAG- and collagen-mimetic peptide nanofibers on the underlying mechanisms of biomineralization through in vitro and in vivo models.

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Cartilage Tissue Regeneration

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Joint defects may originate from injuries, infectious diseases or congenital disorders, and are widespread among children and adults alike. While clinical treatments may partially restore joint mobility and alleviate certain symptoms associated with cartilage loss, post-treatment prognoses are generally poor and the damaged joint can seldom regain its original function. Treatment efforts are complicated by the low regenerative capacity of cartilage, which stems from the absence of blood vessels, lymph nodes and nerve fibers in this tissue, as well as the general scarcity of cells within the cartilaginous matrix. However, as the cartilage extracellular matrix is essential for the repair of osseous and cartilaginous tissues, a biomimetic protein matrix capable of serving a similar function is promising for the regeneration of cartilage. Our objective is to overcome the difficulties associated with the treatment of cartilage defects by employing a biomimetic tissue engineering approach for the stimulation of cartilage regeneration. For this purpose, we design and synthesize self-assembling protein amphiphiles to imitate nanoscale architecture of the cartilage extracellular matrix, and functionalize these peptide assemblies with bioactive groups to enhance the natural regeneration of cartilage tissue. Using these nanofibers, we aim to produce a biomimetic scaffold capable of directing cell growth and differentiation pathways for the optimal repair of cartilaginous tissue.

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Wound Healing

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Wound healing is an intricate process that involves not only the repair of an organ or tissue following injury, but also the mechanisms by which the wound site is protected and the newly dividing cells are directed during the regeneration process. Despite the prevalence of injuries involving osseous and cartilaginous tissues, wounds in these regions are exceedingly slow to heal. In addition, even rapidly healing tissues, such as skin, can suffer from impaired regenerative capacity in individuals with disorders such as diabetes. Consequently, increasing the natural regenerative capacity of the human body is of substantial importance, and peptide amphiphiles have received considerable attention as potential agents for the hastening of the wound healing process. Our laboratory utilizes heparin-mimetic peptide amphiphiles in the regeneration of diabetic ulcers and acute wounds using a rat model. In addition, we also use a laminin-mimetic peptide (lauryl-YIGSR) to facilitate the healing of an artificially damaged cornea in a rabbit model. These peptide amphiphiles can potentially be used as topical remedies for a variety of skin and eye injuries, and are particularly advantageous in that they are easy to apply and unlikely to cause immune complications.

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Molecular Characterization of

Movement Disorders

 

Increasing life expectancies around the globe continue to raise the incidence of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), essential tremor (ETM), and spinocerebellar ataxia (SCA), necessitating the development of effective treatment procedures for these slow-acting but severely debilitating conditions. The identification of molecular factors underlying such disorders is crucial for the development of novel diagnostic and therapeutic approaches, and recent breakthroughs in next generation sequencing techniques and genome-wide association studies have revealed several causative mutations and risk factors in this context. However, many other causative elements are yet to be identified, and a combination of genetic and molecular approaches is necessary to fully elucidate the pathways by which mutations, epigenetic factors and the external environment can trigger the onset of disease. Our laboratory is working on the identification and characterization of genes responsible for causing hereditary neurological disorders. We combine next-generation sequencing techniques with molecular functional studies in order to identify new disease-associated genes, as well as to understand the role of previously identified genes on the pathogenesis of this disease.

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Drug Delivery and Nanotoxicology

 

Drug Delivery Platforms

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A great majority of drugs fail to pass through clinical trials due to detrimental side effects or short plasma half lives, and drug nanocarrier systems have received considerable attention in the recent decade as target-specific vectors capable of avoiding such complications. The main focus of such approaches is to develop a nanocarrier system by which a therapeutic molecule can be delivered selectively to its target tissue or cell type, hence increasing drug efficacy, reducing effective doses and minimizing toxic effects compared to the administration of the vector-free drug. Our group studies a wide array of drug vectors, including peptide amphiphile nanofibers, nanospheres, liposomes and SPIONs (superparamagnetic iron oxide nanoparticles). We have previously demonstrated that a cationic peptide amphiphile (lauryl-VVAGK) could serve as an efficient vector for the delivery and slow release of therapeutic oligonucleotides, (Bulut, 2011) and are currently investigating different strategies for the enhacement of drug uptake and targeting capabilities in liposomes. In addition to conventional drugs, we are also working on the peptide amphiphile-mediated delivery of SPIONs, novel MRI contrast agents with low toxicity and considerable potential in theranostics, and have previously shown that SPION solubilities were improved following non-covalent functionalization by the peptide amphiphiles lauryl-VVAGK and lauryl-VVAAD (Sulek, 2011).

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Nanotoxicology

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Advances in nanotechnology have resulted in the development of novel engineered nanomaterials with different physicochemical properties for numerous applications in the electronic, chemical, biological and medical fields. Engineered metal nanoparticles (ENPs) are among the most widely studied nanomaterials used in biomedical applications, including biosensors, targeted drug and gene delivery, tracking and theranostics. Our group also uses peptide amphiphile structures, supermagnetic iron oxide nanoparticles (SPIONs) and gold nanoparticles for drug delivery and imaging purposes; however, the environmental impact of these nanoparticles are not fully detailed in the literature. As such, we are currently focusing on determining the toxic effects of these materials on various cell lines. In particular, we are using gold nanoparticles as a case study to illustrate the importance of the biological milieu (i.e. the environment that a nanoparticle experiences within an organism) on the uptake, transport and toxicity mechanisms of nanomaterials.

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