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DIFFERENTIATION OF HUMAN ENDOCHONDRAL CARTILAGE CULTURED IN THE HYDRODYNAMIC FOCUSING BIOREACTOR.  D. Montufar-Solis¹, J. Hecht² and P.J. Duke¹. ¹Dept. of Orthodontics, Dental Branch, ²Dept. of Pediatrics, Medical School, University of Texas Health Science Center at Houston, Houston, TX.

   We previously demonstrated that the slow turning lateral (STLV) and the high aspect ratio (HARV) vessel bioreactors provide a breakthrough in the way the systems support growth and differentiation of endochondral cartilage.  Mouse cartilage can be grown from undifferentiated limbbud cells and such cartilage is suitable for healing of defects in membranous bone.  Human chondrocytes expanded in monolayer culture stop expressing the cartilage phenotype, but upon subsequent culture in these bioreactors, the cartilage phenotype is expressed.  Large cartilaginous nodules with regions of hypertrophy and typical cartilage matrix are formed.  The objective of the present study was to assess the capability of the hydrodynamic focusing bioreactor (HFB) to support growth and differentiation of human endochondral cartilage.  A single cell suspension of costochondral chondrocytes was allowed to aggregate over night and aggregates were cultured in the HFB for 25 days.  The aggregates stayed mostly towards the center of the vessel and after 5 days of culture had fused into one large nodule.  Rotation was adjusted during culture from 17-30 rpm, an indication of the change in size/mass of the nodule.  The large spheroid was fixed, sectioned, and stained.  The extracellular matrix stained positively for collagen II and appeared histologically similar to that seen before, but the metachromatic staining that results from toluidine blue interacting with the glycosamynoglycans (GAGs) of cartilage matrix did not occur in these cultures.  Production, secretion and aggregation of GAGs in cartilage are affected by mechanical force, so these results indicate that the chondrocytes in the HFB did not receive sufficient mechanical stimulation, possibly due to their continued state of suspension in the medium, and that the HFB bioreactor may approach closer a spaceflight environment than previously tested bioreactor vessels in terms of load experienced by the cells.

(NASA-JSC Cooperative Agreement #NCC9-142 to PJ Duke, Shriners Hospital for Children grant #15955-JTH.)