The objective of this research is to translate previous work on flexor tendon tissue engineering in the rabbit model to human clinical cases. Specifically, the goals &objectives are to: 1) Optimize techniques for acellularization of human flexor tendons based on work in the rabbit;2) Seed acellularized human tendons with candidate cells to create tissue-engineered tendon constructs;3) Maximize tissue-engineered tendon construct strength and viability in vitro by applying cyclic shear and strain forces using a novel large-scale tissue bioreactor;and 4) Translate to select human clinical cases by using these tissue-engineered tendon constructs for tendon reconstruction in severe cases of mutilating hand injuries. Human flexor tendons will be dissected and preserved in culture. Conditions using SDS, Triton x-100, and freeze-thaw cycles will be varied until the optimal protocol to minimize antigenicity while maintaining structural integrity is established. Effectiveness will be determined by histology, fluorescent cytostaining, and DNA content. Structural strength will be determined by tensiometry for ultimate tensile strength and elastic modulus. Primary cultures of tenocytes, dermal fibroblasts, and adipoderived stem cells will be expanded in culture and seeded at a density of 2x106 cells/cc. Seeding will consist of combinations of cell suspension, microinjection, and ultrasonication. The tendon constructs will be kept in culture for 7 days. Cell seeding efficacy will be determined by H&E microscopy, cytostaining, and quantitative analysis of collagen I &III. A custom tissue bioreactor providing uniaxial tendon strain will be used. The tendon constructs will be subjected to a uniaxial stretch force 1.25N each over a 5 day course. The initial cycle parameters will be 1cycle/min in alternating 1 hour periods of mechanical loading and rest. After bioreactor treatment or stationary incubation, the specimens will undergo tensile testing to compare ultimate tensile stress and elastic modulus. In Veteran patients with severe upper extremity injuries, both sets of flexor tendons are missing [flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS)]. The FDS tendons are a redundant system that is usually not reconstructed. This provides a unique opportunity to test efficacy of the tissue-engineered tendon grafts with minimal additional operative time and risk to the patient. The FDPs will be reconstructed using normal tendon grafts and the FDSs will be reconstructed using the new tissue-engineered tendon grafts. Outcomes will consist of postoperative range of motion, histology on biopsies, and need for revision surgery. After these techniques are developed, VA surgeons could remove a small portion of tendon (or other cell source) and then allow cells to proliferate in culture while the patient is stabilized. Then, cadaver allograft tendons from a tissue bank can be acellularized and seeded with the patient's own cells. When reconstruction of the extremity is undertaken, large amounts of biocompatible tendon would be available. This bioengineering research may be translated to direct clinical applications for a significant need in injured soldiers.

Public Health Relevance

This translational research will optimize techniques to produce tissue engineered tendons. After these techniques are developed, military and VA surgeons could remove a small portion of remaining tendon (or other cell source) and then allow these cells to proliferate in tissue culture while the patient is stabilized. Then, cadaver allograft tendons from a human tissue bank can be acellularized and seeded with the patient's own cells. When definitive reconstruction of the extremity is undertaken, large amounts of biocompatible tendon material would be available. Therefore, recent advances in bioengineering research may be translated to direct clinical applications for a unique and significant need in injured soldiers. Furthermore, aging veterans have increased incidence of rheumatoid arthritis and osteoarthritis which lead to degeneration of tendons and decreased hand function. Tissue engineered flexor tendon grafts would also be useful for reconstruction in these cases.

Agency
National Institute of Health (NIH)
Type
Non-HHS Research Projects (I01)
Project #
5I01RX000275-03
Application #
8838111
Study Section
Translational Rehab (Basic) (RRD0)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Veterans Admin Palo Alto Health Care Sys
Department
Type
DUNS #
City
Palo Alto
State
CA
Country
United States
Zip Code
94304