3D Gels for Pericardial Cell Engineering
Gundu H. R. Rao, Thomas Chandy
Laboratory Medicine and Pathology
University of Minnesota
A) Specific Aims
Hydrogels are providing new opportunities for a variety of medical applications. The aim of this research project is to develop and investigate the potential of stem cell delivery through three-dimensional composite biodegradable matrix of gelatin/chitosan gels formed via in situ enzymatic cross linking to mimic the natural pericardia. The main objectives include (a) To develop a composite 3D-gel matrix based on water soluble gelatin/chitosan via enzymatic crosslinking techniques, (b) To tissue engineer the gel scaffolds with porcine bone marrow stem cells via seeding the cells during the gelation process and (c) To investigate the viability of the cells for regeneration in in vitro simulated conditions and observe the cell proliferation and the extra cellular matrix formation in the gel scaffold.
The hypothesis is that the cells grown in a three-dimensional bioresorbable scaffold would be gradually replaced by new cells to form functional tissues. It is suggested that the self-assembly of these novel composite erodable matrix (gelatin/chitosan) can produce a complex hierarchical structure (open porous channels and mechanical stability) for developing a three dimensional scaffold for cell attachment and growth. This bone marrow-derived stem cells may be used for regenerating the pericardial tissues for naturalization and a cure for congestive heart failure.
B) Background and Significance
Tissue loss or organ failure is one of the most tragic as well as costly problems in human health care. It has been estimated that annual US health care costs associated with tissue loss or organ failure exceed 400 billion dollars (1,2). The functional deficits caused by tissue and organ failure can at present be only partly ameliorated by transplantation surgery or the use of mechanical devices. However, transplantation is currently limited due to shortage of donor tissues and organs. Tissue engineering is an interdisciplinary field that incorporates principles of engineering and polymer chemistry into biological sciences, in efforts to develop biological substitutes for failed tissues and organs (1-3). Hence, there is an urgent need for tissue-engineered alternatives to reconstruct the failing tissues and organs.
Tissue engineering techniques generally require the use of a porous, bioresorbable scaffold, which serves as a three-dimensional template for initial cell attachment and subsequent tissue formation, both in vitro and in vivo. The ability of the scaffold to be metabolized by the body allows it to be gradually replaced by new cells to form functional tissues. Many important factors are involved in the design of systems to culture and grow functional tissues in the laboratory (2,4). Among them are: (1) be highly porous with an interconnected pore network for cell growth and flow transport of nutrients and metabolic waste; (2) be biocompatible and bioresorbable to match tissue regeneration; (3) have suitable surface chemistry for cell attachment, proliferation and differentiation; and (4) have excellent mechanical properties. Both synthetic and natural materials have been served as exogenous three-dimensional scaffold for tissue development. These biomaterials include poly glycolic acid, polylactic acid, polylactic-c0-glycolic acid (5,6), calcium phosphate ceramics (7), collagen, alginate, hyaluronate and laminin (8-10).
Several extracellular matrices (ECM) –like materials that combine synthetic polymer with three-dimensional collagen gels have been investigated. For example, polyethylene glycol/collagen composites have been used as in vivo scaffolds for connective tissue regeneration (11), whereas fibronectin-collagen and laminin-collagen composites have been used to grow cells in vitro (12). Tan et al (13) have shown that a hybrid chitosan-collagen matrix has potential biological and mechanical benefits for use as a cellular scaffold. Cell adhesion molecules (eg, RGD, fibronectin, growth factors) have been evaluated extensively for surface modification to enhance cell adhesion or to allow biospecific cell adhesion. Many cell types have been successfully cultured on these scaffolds, including smooth muscle cells, endothelial cells, hepatocytes and chondrocytes (3,8,10). However, tissue engineering with stem cells and their use in in situ pericardial reconstructions are limited. Thus, there is an urgent need to search for novel methods of generating pericardium and their use to repair the failing heart muscles.
There are few common approaches for creating gels that could be extended to in situ systems. One approach commonly used for in vitro gel formation is to initiate polymerization reactions in the presence of multi-functional monomers, like cyanoacrylate adhesives (14). Since, low-molecular weight and reactive monomers are used, this approach raises concerns of toxicity and compatibility. A second approach for forming gels is to use “smart” polymers that gel in response to the environment conditions (temperature, pH etc) after injection. Typical smart polymers are gelatin, poly(ethylene oxide)-poly (propylene oxide) polymers (15,16). A third approach for gel formation is to crosslink linear polymers, monomers or macromonomers using light or chemical agents (17). However, biocompatibility of the products are still a concern. Recent studies of Chen et al (18) have indicated that a microbial transglutaminase and a mushroom tyrosinase are capable of in situ gelation of gelatin/chitosan solutions. Both biopolymers have been reported to be biocompatible and non-immunogenic (18, 19).
Until recently, it was thought that tissue specific stem cells could differentiate into cells of only that type of tissue. However, a number of recent publications have suggested that adult organ specific stem cells may be capable of differentiating into cells of different tissues (20-23). Donald et al (24) have shown that bone marrow stem cells infused at the time of a coronary occlusion can 9 days later differentiate into myocytes and smooth muscle cells. This could be considered within the realm of possible differentiation potential of mesenchymal cells that are present in marrow. Then, why does stem cell plasticity exist, and what role do cells with this characteristic have in vivo? Most studies showing differentiation into cell types outside the normal differentiation process have shown that this occurs almost exclusively in organs that have been damaged: ischemia for endothelial engraftment (20), cirrhosis for liver and bile duct engraftment (21), toxin administration (22) or muscular dystrophy (23) for muscle engraftment, or when the organ is growing (contribution of human CD34+ cells to liver when transplanted in pre-immune fetal recipients. Thus, such stem cells may have as mission to repair tissue damage.
This study explores the possibility of developing techniques for the gelation of novel hydrogels of gelatin and chitosan through enzymatic reactions, to form 3D matrices for stem cell delivery. Selected hydrogel combinations will be utilized for stem cell seeding and will be modified with bioactive peptides (like RGD peptide) for mimicking natural extracellular matrix (ECM) for generating the in situ pericardia. Stem cells will be seeded homogeneously in the hydrogels during enzymatic cross linking remain viable, proliferate and produce ECM proteins. It is assumed that the stem cell seeded gelatin/chitosan gels formed via in situ enzymatic cross linking will mimic the natural pericardia, when injected intrapericardially to the dilated hearts.
C. Preliminary Studies
This research project combines the expertise and the research strength of different research groups from cardiovascular division, lab medicine and pathology of University of Minnesota and from a small business company . The background of each of these researchers is complementary, which enhances the execution of this multidisciplinary research program. The principal investigator (Dr. Gundu Rao) has been active in the area of platelet biochemistry, pharmacology, biomaterials, cardiovascular device development and research for several years. The principal investigator’s group have been active in the areas of biomaterial surface characterization, modification and development of targeted drug delivery systems, development of biodegradable polymer matrix components, studies on blood compatibility of modified biomaterial surfaces and medical devices and testing newly developed cardiovascular devices in animal models (25-28).
Previous studies from our group have shown that polyethylene glycol grafted pericardial tissue is resistant to calcification, platelet adhesion and degradation (25). Drug encapsulation and delivery through polymeric microstructures has been one of the fields of our interest. We have focused our efforts on developing drug delivery systems with improved biocompatibility and controlled delivery for the therapeutic cardiovascular applications (29). In addition, we have used a mild chitosan/PEG/calcium alginate micro encapsulation process for encapsulation of macromoleculs (such as albumin, insulin and hirudin) and cells (RBC) for therapeutics (27). Recently, we have developed a spray coating method (28) for fabricating hollow tube structures of a polylactic acid/poly ethylene vinyl acetate blend; to be used for nerve guided regeneration (NGR).
We have done some work in developing a series of three-dimensional matrix from Alginate/elastin/PEG and PLA/PEVAc blends. Scanning electron micrographs of Alg/Ela/PEG and PLA/PEVAc membranes are shown in figure 1. The surface
morphology (Fig 1A) and ultra structure (Fig 1B) of Alg/Ela/PEG membrane demonstrates macro porous and open channels (35- 100 mm). The PLA (Fig 1C) and
Fig 1: SEM micrographs of (A) Alg/Ela/PEG composite matrix surface, (B) it’s ultra structure, (C) PLA matrix surface and (D) PLA/PEVAc blend membrane surface.
PLA/PEVAc (Fig 1D) membranes have also indicated porous surface structures (~ 50-125mm). This porous scaffold architecture is suitable for cell integration and formation of structured and functional tissue interfaces.
The collaborating investigator (Prof. Das) has designed and developed a new superelastic Nitinol-Dacron, Double-disk self centering atrial septal defect closure device and studied its efficacy in a canine model of atrial septal defect(30). This novel device is successfully being implanted in humans, for closure of atrial septal defects. This device developed at the University has been licensed to a medical devise company in St. Paul (Microvena Corporation).
Another collaborating investigator (Prof. Zhang) has experiences in pig bone marrow stem cell selection, expansion and characterization. Selection of multi-potent progenitor cell (MAPC) from swine marrow was initiated with the goal of obtaining autologous cells for transplantation. Bone marrow aspirate was collected from Yorkshire pigs (n=8), and cells were depleted of CD45 and red blood cell lineage cells using primary antibodies against pig CD45 and pig red blood cells, and secondary antibodies attached to magnetic microbeads (Miltenyi Biotech). The cells were plated and cultured under the conditions of fibronectin coated plates, with 10 ng/ml EGF and PDGF-BB. Attached cells begin growing in approximately one to four weeks. The cells appear to grow well in culture conditions, and have doubling times 24~36 hours. At present time the sMAPC have undergone > 40 cell doublings, and is continuing. The cells are HLA-DR negative. We are currently evaluating other molecular markers, as well as the ability of these cells to differentiate into different lineages, particularly cardiomyocytes, vascular smooth muscle, and endothelial cells. Swine MAPC are transfected with markers to help in identification using replication deficient retroviral vectors containing plasmid construct encoding for green fluorescent protein (GFP) and beta galactosidase (X-Gal) enzyme. Figure 1 shows one cell line of sMAPC labeled with beta galactosidase (X-Gal) enzyme or green fluorescent protein (GFP) (right).
We believe that the interdisciplinary nature of this project would be an ideal model for medical research, biomedical engineering and product development. The techniques of gel matrix development and their evaluation has been established in our group. Bone marrow stem cell culture and their growth have been established in petri dishes in Prof. Zhang’s laboratory. Clearly, we have the capability to carry out this project and the initial groundwork has been firmly established.
D. Research design and methods
i) Fabrication of three-dimensional gelatin/chitosan gel matrices
The concept of the preparation method is based on a previously published protocol for gelatin/chitosan gels (18). Typically, a concentrated gelatin solution (10w/v%) will be dissolved in deionised water and the pH adjusted to 6.0 using small amount of 1M NaOH. A concentrated chitosan solution (1.6w/v%) will also be prepared in 0.1M HCl, pH 2. After stirring overnight, the insoluble particles will be removed by filtration. The pH of the chitosan solution will be adjusted to 5.9 to 6.0 with 0.1N NaOH. Gel formation will be initiated by adding enzyme (tyrosinase 60U/ml or transglutaminase 10 U/g-gelatin) to solutions containing gelatin or blends of gelatin and chitosan. A series of gelatin and chitosan blends will be used for obtaining the optimum gels for stem cell growing.
ii) Preparation of stem cell loaded gelatin/chitosan gel matrix
a) stem cell culture and cell detection methods etc..
We will use marrow from swine to generate swine multi-potent progenitor cells (sMAPC). In initial experiments, marrow will be Ficoll-Hypaque separated and plated under human or murine MAPC culture conditions. Cultured cells will be examined for cell surface antigen expression and expression of primitive markers at the RNA level as described in the introduction to specific aims. As discussed in SA1 for human cells, this knowledge will be used to further purify MAPC from marrow. Cultures will be examined for cell expansion capacity (cell doubling number, telomerase activity and telomere length) and differentiation potential. The stem cell lab keeps the stem cell cultures and this cells will be used for this project as well.
b) Interpretation/potential problems
Marrow stem cells have been generated from marrow of mice, sheep and primates. However, we have shown that MAPC can be generated from murine marrow with minor modifications to the methods used for human multi-potent progenitor cells (hMAPC). Whether conditions used for these species or humans will be transferable to the swine model is not yet known.
c) Culture conditions: As we did for mMAPC, we will test generation of sMAPC by plating marrow as a mononuclear cell population in hMAPC or mMAPC culture conditions. Once cells with the morphology of MAPC are detected, cells will be harvested and depleted of CD45+ cells. We have obtained an anti-swine CD45 Ab. As described for mMAPC and hMAPC in preliminary studies, we will test different approaches to optimize culture conditions for sMAPC. We will test the role of additional cytokines or ECM components identified to be important for hMAPC and mMAPC growth. As all cytokines used will be of human and the effect of human cytokines on sMAPC may be sub-optimal, we will test murine cytokines, when available, or increase the concentration of the human cytokines to find the optimal concentration.
d) stem cell loaded gels