Projects

Synthesis of Antimicrobial Surfaces

Enzyme Therapy for Mitigating the Effects of Scarring

In vitro Folliculogenesis: Maturation of Ovarian Follicles

Plasma Modified Surfaces for Tissue Engineering Applications

Synthesis of Antimicrobial Surfaces:

A survey of biocidal surfaces shows that most current technologies have serious shortcomings when the goal is the preparation of permanently biocidal surfaces.  These shortcomings include low levels of biocidal activity, release and loss of the biocidal component from the surface, short half-lives of the biocidal agent, and use of harsh application methods.   We are working to overcome these shortcomings by the discovery and testing of new materials and processes that can produce long-lasting antimicrobial surfaces.  We are currently using several approaches towards this end.

One approach is the synthesis of surface active polymeric compounds.  These materials are either synthesized in solution and then attached to surfaces or synthesized in situ by chemical or physical polymerization at a surface.  Our primary target compounds in this work are polyquaternary amines but we are also interested in compounds in the halamine group and in complex materials that may include combinations of surface bound and diffusible antimicrobial materials.

Compounds on or in materials that contact the environment and which can produce persistent antimicrobial activity are sought in a number of different areas including the defense/homeland security industry - for protection against biological attack; the building industry - to fight mold infestation; the food industry - to reduce the bacterial load on food preparation surfaces; the packaging industry - to provide sterile containers; and numerous industries for reduction of biological load due to biofilms. 

The effect of anti-microbial surfaces on the prevention or inhibition of biofilms is important in many areas. 
Our current interests are in the prevention of biofilms in indwelling medical devices such as catheters. 
Prevention of biofilm formation in this area would reduce the number of catheter related infections resulting in better medical outcomes and lowered treatment costs.

~Questions about this project, please contact Rick Koepsel at 412-235-5126 or rrk1@pitt.edu

Enzyme Therapy for Mitigating the Effects of Scarring:

Reversing the formation of fibrotic (i.e. scar) tissue in skeletal muscle, which prevents complete regeneration and recovery of function, remains a critical challenge for clinicians and tissue engineering researchers. The fibrous collagen matrix, which beings forming approximately two weeks post-injury, inhibits progenitor cells from infiltrating the site of injury where they can differentiate into new tissue. Although several therapies, including the use of growth factors and drugs that inhibit the fibrosis pathway, have met with clinical success, existing treatments are based on scar prevention and thus must be administered within a short timeframe after injury to be effective.

A therapy that degrades scar tissue during or after completion of the natural remodeling process would represent a marked improvement in regenerative medicine. To this end, we are investigating the therapeutic benefit associated with administering matrix metalloproteinases (MMPs), proteolytic enzymes that catalyze the degradation of collagens, to pre-existing scar tissue. The exogenous enzymes may be delivered to the site of injury via direct injection or controlled release from a biomaterial such as a suture. Chemical modification is being explored as a means to stabilize the therapeutic MMPs, thereby, prolonging their effective half-life.  Regeneration of functional tissue will ultimately reduce rehabilitation time and frequency of re-injury.

~Questions about this project, please contact Joel Kaar at 412-235-5208 or at kaarjl@upmc.edu

In vitro Folliculogenesis: Maturation of Ovarian Follicles:

Therapeutic treatments for cancer and other diseases often adversely affect the reproductive cells of a patient. The very therapy that kills the cancer cells also kills the germ cells within the reproductive organs. For girls and women, this is a particularly harsh consequence because mammalian females are born with all the eggs they will ever have. For adult women, depletion of the supply of eggs results in menopause. Children whose eggs have been destroyed fail to undergo puberty. To a young girl, facing lifetime sterility as a result being cured of her cancer, a technique that would allow her to bear her own genetic children would be welcome indeed. One step that is being taken is to remove and freeze the ovaries of girls and women prior to their undergoing potentially sterilizing therapies. Unfortunately this process kills all but the most immature follicles. A method to produce mature eggs from immature follicle would restore fertility. 
Such a method is the subject of this proposal in which we use the rat as a model organism. The aims of this project are to use tissue culture engineering approaches to induce the maturation rat follicles. Protocols for culture manipulation and media augmentation are investigated. New and rationally designed bioreactors are being developed. The progress of the maturing oocytes is monitored by gene expression assays on gene chips and with PCR, and in vitro fertilization methods are being optimized.

Another aim of the project looks at the quality of the fertilized oocytes by growing them into embryos and isolating embryonic stem cell lines. The efficiency of this process and the quality of the derived cell lines are indicators of the quality of the in vitro matured eggs. Further proof that the in vitro treatments do not adversely affect the eggs will be obtained from implantation of in vitro derived embryos and the production of pups.  A final set of investigations will determine the whether or not the stem cell lines derived from in vitro matured oocytes can produce clinically relevant tissues. Stem cells lines will be forced to differentiate down the hepatic lineage. These cells will be characterized and then used in a rat liver damage model to demonstrate that they are fully competent for use in regenerative medicine protocols.

The development of a successful follicle culture system will allow the dignity of fertility after cancer for a substantial number of girls and women. In addition, a more efficient source of matured oocytes will alleviate the need for the uncomfortable and potentially risky procedures involved in egg donation. By using an in vitro maturation system, derivation of embryonic stem cell lines could be achieved by maturing follicles obtained from tissue normally discarded after surgical removal or from banked ovarian tissue rather that through direct donation.

~Questions about this project, please contact Matt Heise at 412-235-5208 or at  mkh5@pitt.edu

Plasma Modified Surfaces for Tissue Engineering Applications

Plasma, the fourth state of matter, is a highly energized gas that is used to modify surfaces in many industrial processes.  Industrial plasmas, however, are often hot plasmas with temperatures exceeding 1200 °C. With such extreme temperatures, the use of these plasmas is often limited to metals. Dow Corning Plasma Solutions has developed an atmospheric pressure plasma deposition system which generates a “cold” plasma in the range of 40 – 60 ºC.  We have been using this system to modify the surface of tissue culture polystyrene.

One specific modification that we have been exploring is the permanent attachment of poly-N-isopropylacrylamide (NIPAAm) to culture dishes.  This temperature sensitive polymer has successfully been attached to polystyrene by other surface modification techniques.  It’s attachment to the surface allows for the removal of cells by changing the surface hydrophobicity with temperature; at temperatures above 32 °C the polymer is hydrophobic and below 32 °C it is hydrophilic. Harvesting cells using cultureware treated in this manner is done simply and without degradative enzymes.  In fact, the cells can often be removed intact as cell sheets.

Other applications we have been looking into include surface patterning. Modifications by plasma deposition are precisely controlled by a computer. This will enable us to create patterned surfaces using a desired combination of biologically relevant materials. Further, we are exploring the possibility of using the plasma to make direct modifications to cell surfaces.

~ Questions about this project, please contact Sara Wargo at 412-235-5208 or at slw6@pitt.edu

Updated: February 22, 2007