1) Determining the best cell sources and scaffold materials for TEHV development.

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1 Broadly speaking, my primary research interests focus on the development and application of methodologies that can be employed in the basic understanding and optimization of tissue engineered heart valves (TEHVs). I also have a secondary interest in cartilage tissue engineering (TE). Specifically, I am focusing on the following themes: 1) Determining the best cell sources and scaffold materials for TEHV development. 2) The study of biomechanical and/or biochemical events that aid in engineered heart valve tissue formation. 3) The development/use of magnetic resonance imaging (MRI) and cellular MRI methods to provide noninvasive and nondestructive monitoring/assessment of engineered cardiovascular/musculoskeletal tissue and the cells from which they are derived, particularly when translating to in vivo models and clinical systems. 4) Computational fluid dynamic (CFD) models development to predict the effects of fluidinduced shear stresses on tissue formation. My research plan for the next 5 years will be an effort to comprehensively and hopefully conclusively examine the following: 1) I am very interested in studying the co-culture effects of endothelial progenitor cells (EPC) with bone-marrow derived mesenchymal stem cells (BMSCs) for tissue engineered heart valve (TEHV) development. Reports have focused on the use of either EPCs or BMSCs in heart valve tissue engineering [1]. The reality is that eventually, two cell types will be required to match native heart valve tissue morphology. These are the endothelial (ECs) and myofibroblast (MYFs) cell types. While EPCs have demonstrated capabilities of differentiating to ECs [2] and BMSCs to smooth muscle cell lineages [3], no conclusive evidence has shown a single adult progenitor cell type capable of differentiating simultaneously to both phenotypes. It should be noted however that both cell types are readily accessible and show clinical relevance; EPCs through blood withdrawal and BMSCs through the aspirate of bone marrow. My interests in TEHVs has led me to believe that experiments involving co-culture experiments of and related optimization of the culturing conditioning conditions through biochemical (e.g. growth

2 factors) and biomechanical (fluid-induced shear stresses) cues is one of the keys to TEHV development. As an alternative cell source, I am also interested in exploring the possibility of utilizing human umbilical cord-derived progenitor cells (huc-dpc) as a single cell source capable of concomitant EC and MYF differentiation [4]. I am interested in utilizing Electrospun poly(l-lactide-co-ε-caprolactone) (P(LLA-CL) and electrospun collagen, separately, or in combination for TEHV development. Studies from the Ramakrishna laboratories [5-6] in Singapore have demonstrated the use of these scaffold materials to be particularly suited for smooth muscle cell and endothelial cell proliferation. I propose to utilize these types of scaffolds in the TEHV development effort. Another scaffold of investigation with the aforementioned co-culture experiments (EPCs+ BMSCs) would be the incorporation of glyconsaminoglycan (GAGs) into collagen nanofibrous scaffolds. The four scaffolds to be investigated would therefore be as follows: i) electrospun P(LLA-CL), ii) electrospun collagen, iii) combination of i) and ii) and iv) combination of ii) with GAGs. 2) I am in the process of designing a flow induced-stretch-flexure stress (FSF) bioreactor device that is capable of mimicking the physiological scales of these stresses. The three stress states can be de-coupled and as such the use of this device will be useful in examining the effects of individual and coupled stresses on tissue formation in cardiovascular applications such as in TEHVs and in blood vessel development. Simultaneously I wish to incorporate additional experiments using this system via several combinations of serum, growth factors and cell sources. In this manner, this device will act as a platform for the optimization of cardiovascular tissue constructs. 3) I hypothesize that the importance of noninvasive and nondestructive monitoring of the cellular function within the developing valvular tissue is a critical aspect of implant success. I thus propose an in-depth study on the longitudinal (temporal) position and migration patterns of cells during the tissue development process. This can be achieved through cmri techniques such as with the labeling of cells with superparamagnetic iron oxide (SPIO) particles. The specific population of cells will be established through the methods detailed in 1) and 2) above. I wish to in particular conduct efficient, non-toxic, endosomal uptake studies of SPIO particles in BMSCs, huc-dpcs, ECs and MYFs with a particular focus on extracellular matrix (ECM) production and differentiation capacity of these cell types after labeling. The point to which these cell types, following SPIO

3 labeling with appropriate concentrations of SPIO particles, remain viable and nonapoptotic, proliferate and if applicable differentiate to the preferred cell lineages, and produce ECM in comparison to unlabeled controls will be determined. I have established previous experience in this area with respect to the SPIO-labeling of chondrocytes for cartilage tissue engineering applications as shown in Figs. 1 and 2 [7] below: 50 µm 50 µm Figure 1: Abundant staining for proteoglycan (left) and collagen (right) derived from, SPIO-labeled chondrocytes in a nondegradable hydrogel scaffold. 500 µm Figure 2: 200 µm MRI slice (left) of SPIO-labeled chondrocytes (seen as dark spots) after 30 days of incubation, in correspondence to a 5 µm histological section (H&E stained) section taken from the same sample, at approximately same slice location. The next step will involve longitudinal cmri evaluation of cellular migration patterns of the cell types in vitro; first, at the scaffold level in static culture and subsequently dynamically under physiologically relevant flow conditions. We will utilize the FSF bioreactor mentioned previously to subject SPIO-labeled cell-seeded scaffolds to below normal, normal and above normal physiologically relevant flow fields and conduct

4 intermittent longitudinal cmri studies. The exact scaffolds to be used will be determined from 1) above. A modified MRI- compatible sample chamber of the FSF bioreactor will make it possible to conduct the cmri experiments. Based on the in vitro studies, in vivo studies in either the porcine or ovine model can take place; this will involve TEHV implantation and longitudinal cmri tracking of cell position and migration In vivo longitudinal cellular distribution and tissue formation would be studied over the first 10 weeks following implantation, the duration in which most valve remodeling would occur. In this way, we would be able to noninvasively and nondestructively track and better understand changes in cell fate during in vivo tissue development. In addition SPIO-labeled cells would be intravenously injected as a means of cell-based therapy to determine if the TEHVs can be supplemented with additional cells during the first few weeks (~ 6 weeks) following implantation. The fate of these cells will also be monitored by cmri. In a similar manner, I am interested in performing studies with cmri, focusing on cell populations involved in tissue engineered blood vessels as well. Finally, I am interested in using MRI methods to monitor extracellular matrix constituents in evolving tissue engineered constructs in vitro, ex vivo and in vivo. One example would be in observing the evolving glycosaminoglycan (GAG) content in tissue engineered cartilage (Fig 3) [8]. Figure 3: Use of an MR-derived parameter (FCD) that correlates well with average GAG content in longitudinally monitored tissue engineered cartilage. 4) I would like to develop CFD models to study the effects of the flow field on TEHV development. Since part of the biomechanical conditioning event may cause samples to move through flexure and/or stretch states, I intend to use moving boundary methods to more accurately predict the flow field based on my previous experience in this area.

5 Preliminary models I have performed in this work suggests that certain sample geometries and certain flow patterns such as oscillatory wall shear stress may be more conducive to tissue formation (see Fig. 4 [9]). Moving boundary methods assume a priori knowledge of the sample motion and generally with TEHV leaflets, the sample motion can be quantified either by analytical approximations or empirically through highspeed image capture experiments. These CFD models will provide insight into the precise nature of the flow field and how that changes with i) varying geometry ii) dynamic sample motion and iii) nature of the flow (pulsatile versus steady). Accompanying experiments in the FSF bioreactor described in 2) coupled with the CFD results will help identify the specific nature of the fluid-induced stresses that optimizes tissue formation. Subsequently a bioreactor system can be developed to maximize the occurrence of this specific flow environment so as to optimize TEHV formation. Similarly I would also like to develop CFD models to study the effects of fluid-induced stresses on tissue engineered blood vessels. Figure 4: CFD simulations showing high degree of oscillatory shear under flexed states in rectangular strips of tissue engineering scaffolds. References 1] Siepe M., et al., European Journal of Cardio-thoracic Surgery; 34 (2008), ] Cebotari S., et al., Circulation; 114 (2006), I ] Perry TE., et al., Annals of Thoracic Surgery; 75, (2003), ] Schmidt D., et al., Tissue Engineering; 12(11), (2006), ] Mo XM., et al., Biomaterials; 25, (2004), ] Wei H., et al., Tissue Engineering; 11(9/10), (2005), ] Ramaswamy, S., et al., Submitted manuscript undergoing journal review, August ] Ramaswamy S., et al., Tissue Engineering, Part C Methods. (2008), [Epub ahead of print]. 9] Ramaswamy S., et al., 8 th World Biomaterials Congress: Crossing Frontiers in Biomaterials and Regenerative Medicine, Amsterdam, The Netherlands, May 28 th June 1 st 2008.