© 2018 by A. Putnam and the CSET Lab. All rights reserved.

Overview

The field of regenerative medicine has witnessed impressive advances over the past 25-30 years, moving us ever closer to the goal of translating engineered tissue constructs into human patients. However, despite an exponentially-expanding literature documenting advances in biomaterials and stem cell biology, the two biggest factors limiting the clinical applicability of engineered tissues 20 years ago continue to be the biggest hurdles today: tissue function and vascularization.

 

To address these challenges, we are focused on the instructive signals provided to cells from their surroundings via a complex 3D network of proteins and polysaccharides known collectively as the extracellular matrix (ECM). Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the ECM provides chemical information to the cells with which it interacts. Interestingly, evidence from our lab (and many others) suggests that the mechanical properties of the ECM (i.e., its stiffness or compliance) also provide vital instructional cues to cells, rather than just passive mechanical support. As a result, the view of the ECM has come full circle – from one of a purely mechanical nature, to purely chemical, and now mechano-chemical – supporting the notion that both ECM chemistry and mechanics are critical determinants of cell fate and morphogenesis. We are particularly interested in the ECM's roles in both vasculogenesis and angiogenesis, the two processes by which new blood vessels form in the human body.

Word Cloud Representation of Our Research Interests

We use biomaterials as cell culture platforms in order to better understand mechanobiology and the cell-ECM interface in both 2D and 3D, and then apply our fundamental discoveries to rationally design innovative “cell-instructive” materials that elicit specific molecular behaviors and thereby control cell function. Such cell-instructive materials would have enormous implications for tissue engineering and regenerative medicine applications, and allow for new insights into a variety of pathologies in which cell-ECM interactions are disrupted, including cancer and cardiovascular disease.

Ongoing Research Projects

1.) Microenvironmental control of capillary morphogenesis

Using a combination of in vitro and in vivo models, we have discovered that the formation of nascent vasculature is regulated by both the biophysical properties of the ECM and the identity of the supporting stromal cells. Our data suggest that stromal cells of different origins differentially control ECM proteolysis during angiogenic sprouting, and that controlling the rate of ECM breakdown is critical to yield stable, functional vessels. In this project, we are mechanistically investigating how these two critical instructive elements of the local microenvironment (i.e., the stromal cells and the ECM) influence the quantity, functional quality, and stability of new vasculature.

Collaborators: Steve Weiss, MD (UM Life Sciences Institute), Elliot Botvinick, PhD (UC Irvine)

2.) Capillary morphogenesis in cell-instructive biomaterials

We also utilize engineered hydrogel platforms based on poly(ethylene glycol) (PEG) to understand the influence of the ECM microenvironment on capillary morphogenesis.  By fabricating PEG hydrogels with peptide crosslinks differentially susceptible to different proteases, we can tune the degradation kinetics of the ECM and assess the resulting effects on capillary growth and functionality. We have also conjugated PEG to full-length ECM proteins (PEG-collagen, PEG-fibrinogen) to make biosynthetic conjugates that support complex cellular programs in 3D.  By altering features of the PEG (weight fraction, number of arms, MW, etc.), we can alter the bulk mechanical properties of the gels while keeping the biological properties unchanged. These gels enable us to address fundamental questions in vitro, and have also shown great promise as morphogenetic guides to direct vascularization in ischemic tissues in vivo. 

Collaborators:

3.) Modular approaches to revascularize ischemic tissues

Over the past 5-10 years, a number of studies have demonstrated that vasculature formed in bulk gels in vitro can inosculate (connect) with host vasculature in vitro following transplantation.  These pre-vascularization strategies hold great promise to treat ischemic conditions and potentially overcome a critical challenge in the field of tissue engineering.  However, they require an invasive surgery, which in some cases may not be desirable.  In this project, we are making small vascularized modules by embedding endothelial cells and supportive stromal cells in small biomaterial modules (on the order of 250-400 um in diameter) and culturing them for a period of time to allow the cells to self-assemble into primitive vascular networks. The small microtissues can then be injected in a minimally-invasive fashion, thereby jump-starting the formation of microvasculature in vivo.

Collaborators: Jan Stegemann, PhD (UM BME)

4.) Mechanobiological control of cardiac reprogramming

The human heart has a very limited (almost zero) capacity to regenerate following a heart attack, and most efforts in the field of tissue engineering/regenerative medicine are focused on the use of stem cells and tissue constructs to create functional chunks of heart muscle tissue for transplantation. Successful implementation of such an approach requires solving the vascularization challenge, a major focus of the CSET Lab.  But what if it were possible to directly transform scar-forming cardiac fibroblasts into functional, beating cardiac myocytes?  In fact, seminal work by other investigators has shown such an approach can partially restore heart function in mice after a heart attack.  In this project, we are investigating the ECM's mechanobiological control of this transdifferentiation process, using a variety of experimental tools and platforms (i.e., engineered hydrogels, traction force microscopy, micropillar arrays, etc.) to do so.

Collaborators: Jianping Fu, PhD (UM Mechanical Engineering)

5.) Mechanobiology in 3D Engineered Environments

Over the past 12 years, our laboratory has been very interested in the mechanical influence of the ECM on cells in vitro and in vivo.  We've shown that ECM rigidity influences a variety of cell types and cell functions in 2D, a discovery widely supported by many other studies in the literature.  But how do the mechanical properties of the ECM influence cell fate in 3D, where adhesive ligand presentation, proteolytic sensitivity, confinement (cell spreading), and diffusive transport may all somehow be coupled? To investigate this question, we are using PEG-based engineered hydrogels along with microrheology and 3D traction force measurements in order to carefully assess the influence of ECM mechanics on a length scale relevant for individual cells in 3D matrices. Endothelial cells are our most common cell type in this line of investigation, but we are also investigating how adult stem cells and tumor cells respond to ECM mechanics in 3D.

Collaborators: Elliot Botvinick, PhD (UC Irvine)

6.) Reconstituting the Perivascular Niche

An important hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells (NSCs), mesenchymal stem cells (MSCs) from bone marrow, adipose, and other tissues, hematopoietic stem cells (HSCs), and many tumor stem cells.  Using our in vitro models of the microvasculature, we are investigating how the perivascular location of stem cells influences their fate, as well as the underlying mechanisms responsible for this control.  In a recent study, we have shown that a key integrin receptor on MSCs is required for their association with the vascular basement membrane, and that silencing this receptor disrupts the ability of MSCs to stimulate capillary morphogenesis.  

Collaborators: Darnell Kaigler, DDS, PhD (UM Dental School)