Cartilage Tissue Engineering for Joint Repair
Mentor: Roy Aaron, MD
The hypothesis of these studies is that cell-based engineered cartilage biocomposites can be created using a subset of synoviocytes, which can be induced by growth factors to differentiate to chondrocytes, and that there are potential synergistic factors, both biochemical and biophysical. Additionally, that this approach can be optimized to produce a tissue engineered construct with more physiologic biochemical, ultra-structural and mechanical properties.
Much of the first 3 months of the grant cycle were used for the design and installation of a customized hypoxia chamber to replace the smaller unit that was on site when the application was submitted. The system and a new incubator were purchased with non-COBRE funds and have been installed.
Specific Aim 1: Optimizing the Cell Population
The cell population has been isolated by the use of Dynal magnetic beads. The initial purification was performed by removal of the interfering Type A cells using a negative isolation technique. This purified population, which due to the enrichment of Type B fibroblasts, underwent chondrogenesis to a greater degree than did the mixed cell population. We are currently creating an expanded purification, which will include positive isolation steps with mesenchymal stem cell markers such as CD105, CD90, STRO 1 BRIGHT, and Flox 3 or Sox2.
Specific Aim 2: Optimizing Growth Factor Exposure and Oxygen Gradients We have used TGFÃŸ1 under normoxic conditions to stimulate chondrogenic differentiation of our synovially-derived progenitor cells. Other growth factors, notably GDF-5, BMP-7 and TGFÃŸ3 have been reported to be highly chondrogenic but no direct comparison among them has been made, this component of this specific aim is underway.
We have examined dimerized BMPs, 2, 4 and 7 in normoxic cultures and will examine their chondrogenic potential under graded hypoxic conditions. Addtionally, we have undertaken a systematic examination of the qPCR requirements of these studies. We have examined 18S, ÃŸ Actin, GAPdh, HPRT1, RPL32, TUBB and ÃŸ2M. Of these qPCR internal control candidates, only B2M showed no response to hypoxic conditions. Clarifying the need for great care and close examinations of the conditions, when choosing a control for for reliable qPCR data. A manuscript on this subject is in preparation.
Specific Aim 3: Optimizing the Biophysical EnvironmentOxygen gradients are known to regulate embryonic skeletal development, promote the re-differentiation of passaged chondrocytes, and induce chondrocyte-specific gene expression during mesenchymal stem cell (MSC) differentiation. To date, the synergy of oxygen gradients with chondrogenic growth factors in optimized progenitor populations has not been examined. Our optimized progenitor population will be exposed to varying levels of hypoxia to determine their viability and chondrogenic potential in vitro. This specific aim will initiate mechanistic studies of oxygen regulation of chondrogenesis for tissue engineering.
This specific aim has not yet begun.
Specific Aim 4: In vivo Implantation In Specific Aims 1-3, we will have optimized the precursor cell population, growth factor sequence, and one important aspect of the physical environment and expect to have engineered cartilage biocomposites ready for implantation in vivo. Long term durability and integration of the optimized cartilage biocomposites under load will be assessed in a patello-femoral joint animal model. Serial arthroscopy and biomechanical indentation testing will assess the functional properties of the implanted biocomposites. This specific aim, as stated above, will be held in abeyance until additional funding sources are identified.