Tissue-engineered trachea from sheep marrow stromal cells with transforming growth factor beta2 released from biodegradable microspheres in a nude rat recipient.
OBJECTIVE
The purpose of this study was to evaluate the feasibility of using autologous sheep marrow stromal cells cultured onto polyglycolic acid mesh to develop helical engineered cartilage equivalents for a functional tracheal replacement. We also explored the potential benefit of local delivery of transforming growth factor beta 2 with biodegradable gelatin microspheres.


METHODS
Bone marrow was obtained by iliac crest aspiration from 6-month-old sheep and cultured in monolayer for 2 weeks. At confluence, the cells were seeded onto nonwoven polyglycolic acid fiber mesh and cultured in vitro with transforming growth factor beta 2 and insulin-like growth factor 1 for 1 week. Cell-polymer constructs were wrapped around a silicone helical template. Constructs were then coated with microspheres incorporating 0.5 microg transforming growth factor beta 2. The cell-polymer-microsphere structures were then implanted into a nude rat. On removal, glycosaminoglycan content and hydroxyproline were analyzed in both native and tissue-engineered trachea. Histologic sections of both native and tissue-engineered trachea were stained with hematoxylin and eosin, safranin-O, and a monoclonal anti-type II collagen antibody.


RESULTS
Cell-polymer constructs with transforming growth factor beta 2 microspheres formed stiff cartilage de novo in the shape of a helix after 6 weeks. Control constructs lacking transforming growth factor beta 2 microspheres appeared to be much stiffer than typical cartilage, with an apparently mineralized matrix. Tissue-engineered trachea was similar to normal trachea. Histologic data showed the presence of mature cartilage. Glycosaminoglycan and hydroxyproline contents were also similar to native cartilage levels.


CONCLUSIONS
This study demonstrates the feasibility of engineering tracheas with sheep marrow stromal cells as a cell source. Engineering the tracheal equivalents with supplemental transforming growth factor beta 2 seemed to have a positive effect on retaining a cartilaginous phenotype in the newly forming tissue.
Kojima
Koji
K
Laboratory for Tissue Engineering and Regenerative Medicine, Brigham & Women's Hospital, Harvard Medical School, Worcester, MA 02115, USA. kojima@zeus.bwh.harvard.edu
Ignotz
Ronald A
RA
Kushibiki
Toshihiro
T
Tinsley
Kevin W
KW
Tabata
Yasuhiko
Y
Vacanti
Charles A
CA
D020341:Absorbable Implants; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001854:Bone Marrow Cells / Q000378:metabolism* / Q000473:pathology; D002356:Cartilage / Q000378:metabolism / Q000473:pathology; D024043:Collagen Type II / Q000378:metabolism; D004396:Coloring Agents; D004801:Eosine Yellowish-(YS); D004867:Equipment Design; D005240:Feasibility Studies; D005780:Gelatin / Q000494:pharmacology; D006416:Hematoxylin; D008863:Microspheres; D008955:Models, Cardiovascular; D011100:Polyglycolic Acid / Q000494:pharmacology; D051381:Rats; D011923:Rats, Nude; D012756:Sheep; D017154:Stromal Cells / Q000378:metabolism* / Q000473:pathology; D023822:Tissue Engineering; D014132:Trachea / Q000166:cytology* / Q000187:drug effects / Q000601:surgery*; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000378:metabolism*
D015415:Biomarkers; D024043:Collagen Type II; D004396:Coloring Agents; D016212:Transforming Growth Factor beta; D011100:Polyglycolic Acid; D005780:Gelatin; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin