Dennis Orthopaedics Tissue Engineering Laboratory

Research Foci

ONGOING PROJECTS

Tissue Engineering of Cartilage Subtypes:

This project is aimed at the development of an autologous tissue engineered piece of repair cartilage for addressing the problem of tracheal stenosis in infants. The approach is to investigate the problem in a rabbit model were we combine chondrocytes isolated from different tissue sources, such as ear, nose and joints, and produce biomechanically sound tissue that can be then tested in vivo. This study addresses not only the practical issue of repairing a trachea, but delves into the molecular differences between chondrocytes from different areas of the body. This project is funded through the National Institute of Dental and Craniofacial Research (http://www.nidcr.nih.gov/) at the NIH (R01DE015322).

Total Joint Resurfacing: Using a technology developed in this laboratory to produce large sheets of cartilage (6.5 x 7.5 cm in area), this project is aimed at developing a method to completely replace entire surfaces of joints. Currently the laboratory is developing and testing a new bioreactor for producing cartilage of greater thickness, while working on biocompatible glues to adhere the engineered cartilage to the underlying bone. This program is currently funded by the State of Ohio through the Clinical Tissue Engineering Center (CTEC; http://www.ctecohio.org/).

Engineering of a Neo-Trachea: Using the cartilage sheet technology, we are developing methods to engineer autologous trachea-like tissue that is suitable for use in repairing long segmental defects in a rabbit model. This project is an extension of the Tissue Engineering of Cartilage Subtypes project and preliminary results have been partially funded by the State of Ohio through the Center for Stem Cell and Regenerative Medicine (CSCRM; http://ora.ra.cwru.edu/stemcellcenter/whoweare/whoweare.htm).

The etiology of laryngotracheal and tracheal stenoses is multifaceted, however, long-term intubation or tracheotomy are the most common causes. The traditional procedure for laryngotracheal stenosis has been laryngotracheal reconstruction (LTR) with anterior and/ or posterior cartilage grafting, which has proven to be very successful. We are currently investigating tissue engineered cartilage as an alternative to native cartilage. Various tissue engineering strategies, such as different scaffolds, growth factors and compression culture are researched to fabricate suitable cartilage grafts for laryngotracheal reconstruction.

Laryngotracheal reconstruction with interposition of an anterior engineered cartilage graft. A) An anterior incision was made through the cricoid cartilage and the upper tracheal ring. The engineered graft with perichondrium, harvested from the ear, lies next to laryngofissure. B) The left side of the graft has been sutured to the cricoid and the tracheal ring and is lifted up (TC= thyroid cartilage; TR= tracheal ring; *= cricoid ring)

The treatment of long-segment tracheal stenosis is a very challenging and ongoing problem. A simple tracheal resection followed by an end-to-end anastomosis is often times not advisable and thus, many types of tracheal prostheses have been tried, but with limited success due to immune rejection, graft ischemia and/ or re-stenosis. In an animal model we currently investigate the potential of scaffold-free cartilage to fabricate a vascularized neotrachea.

A vascularised neotrachea was fabricated using scaffold-free engineered cartilage and implanted into the rabbit's abdomen for 10 weeks. The photograph shows a cross-section of a rabbit's native trachea and the engineered neotrachea.

Development of a Targeted Stem Cell: One of the major hurdles for applying stem cells to tissue repair is the efficient delivery of those cells to the tissue of interest. To address this issue, we are developing cellular “paints” that attach themselves to cell membranes and have the capacity to bind either to the matrix components of the tissue needing repair, or to bind to the specific endothelium of the tissue of interest. Our current focus is to define the interactions of “painted” cells on defined molecular substrates, in vitro, and to then apply them for in vivo targeting. The use of targeted cells for the repair of cartilage is funded through the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS; http://www.niams.nih.gov/), and the targeting of cells to endothelium is being funded by the Department of Defense through a grant awarded to Cell Targeting, LLC.

The body has an amazing ability to repair and, in early life, regenerate. Much of this is thought to be performed by Stem Cells. However, in severe injury or disease, these repair mechanisms are insufficient. The goal of this research project is develop a targeted cell so that the cell adheres to sites of injury and repairs the damaged tissue. Two methods of targeting the cells are being investigated - Antibody Directed Cell Therapy (ADCT) and Peptide Directed Cell Therapy (PDCT). In ADCT cells are first coated with palmitated protein G (PPG), a modified recombinant protein that binds to the Fc region of antibodies, then antibodies are used to coat the cells giving a targeted cell.

In PDCT peptides are derived by phage display technology. Phage are genetically engineered to express random peptides on their surface; for a seven amino acid peptide this produces 1.28 x 10^9 different phage. These phage are then selected against the tissue of interest, reducing the phage pool. The reduced phage pool is then amplified and reselected against the tissue of interest. This work has been done in collaboration with Prof. Ruoslahti who had previously developed phage which homed to injured tissue and neovasculature, these peptides were labelled with FITC and injected into mice. Two models have been studied so far: an Ischemic Heart model and an Ischemic Leg model.

A confocal stack animation of a PPG labelled cell coated with FITC-IGG

Heart tissue 24h after Ischemic event, 2h after FITC-peptide injection.

Calf tissue 3 days following ischemic event, 2h after FITC-peptide injection

Clinical Scale Production of Osteoprogenitor Cells: This study, conducted in collaboration with Aastrom Biosciences, Inc. (http://www.aastrom.com/) is focused on optimizing the production of osteogenic cells for their use in a clinical setting. In addition to optimizing growth conditions, we are identifying cell surface markers that are indicative of osteogenic potential and can be used to titer the osteogenic potential of individual cell preparations. These studies are funded through a Small Business Innovation Research (SBIR) grant (R44DK074201-02) from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; http://www2.niddk.nih.gov/).

Bright field image of cube implant

COLLABORATIONS AND PROJECTS UNDER DEVELOPMENT

Microsatellite Instability: A study on the prevalence of markers for microsatellite instability in Mesenchymal Stem Cells is being conducted in collaboration with Dr. Stan Gerson's group in Hematology and Oncology and with Dr. Randall Marcus, Chairman of Orthopaedics.

Lidocaine and chondrocyte survival: Lidocaine is often used as a local anesthetic for autologous chondrocyte transplantation procedures, yet little is know about the effect of lidocaine on the survival, expansion and differentiation of the transplanted cells.

Growth Factor Delivery and Osteogenesis: Methods to improve the extent and rate of osteogenesis repair are being conducted in collaboration with Dr. Eben Alsberg's laboratory (http://bme.case.edu/faculty_staff/alsberg/) in the Department of Biomedical Engineering at Case.

MicroCT Quantification of In Vivo Bone Formation: A method to discriminate between calcium phosphate carrier scaffolds and in vivo fabricated bone in SCID mice is being carried out in collaboration with Dr. Steve Goldstein and the Orthopaedic Research laboratories at the University of Michigan (http://www.orl.med.umich.edu/orl/orl.html).