Phenotype and biological activity of neonatal equine chondrocytes cultured in a three-dimensional fibrin matrix

Dean A. Hendrickson From the Comparative Orthopaedics Laboratory (Hendrickson, Nixon) and the Section of Epidemiology (Erb), Department of Clinical Sciences, and the James A. Baker Institute (Lust), Cornell University, Ithaca, NY 14853.

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 DVM, MS
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Alan J. Nixon From the Comparative Orthopaedics Laboratory (Hendrickson, Nixon) and the Section of Epidemiology (Erb), Department of Clinical Sciences, and the James A. Baker Institute (Lust), Cornell University, Ithaca, NY 14853.

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Hollis N. Erb From the Comparative Orthopaedics Laboratory (Hendrickson, Nixon) and the Section of Epidemiology (Erb), Department of Clinical Sciences, and the James A. Baker Institute (Lust), Cornell University, Ithaca, NY 14853.

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George Lust From the Comparative Orthopaedics Laboratory (Hendrickson, Nixon) and the Section of Epidemiology (Erb), Department of Clinical Sciences, and the James A. Baker Institute (Lust), Cornell University, Ithaca, NY 14853.

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 PhD

Summary

Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 × 105, 1 × 106, and 5 × 106 chondrocytes/ cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, fibroblast-like cells) was statistically higher for implants that received 5 × 106 precultured cells at all time periods than for implants that received 1 × 105 or 1 × 106 precultured cells. The differentiation ratio was statistically higher for implants that received 5 × 106 immediately implanted cells than for other implants at 7 days after implantation. At 14 days, implants that received 5 × 106 precultured chondrocytes had a higher differentiation ratio than did implants that received 5 × 106 chondrocytes that had not been precultured. Among implants that received precultured chondrocytes, total glycosaminoglycan and chondroitin sulfate content was lowest for implants that received only 1 × 105 cells. Among implants that received chondrocytes that had not been precultured, glycosaminoglycan content was not significantly different among the 3 cell concentrations, and chondroitin sulfate content was different only between implants that received 5 × 106 vs 1 × 106 cells. Only after the longest incubation period and at the highest cell concentration studied did preculturing of chondrocytes improve maintenance of phenotype. Preculturing did not appear to influence proteoglycan synthesis.

Summary

Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 × 105, 1 × 106, and 5 × 106 chondrocytes/ cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, fibroblast-like cells) was statistically higher for implants that received 5 × 106 precultured cells at all time periods than for implants that received 1 × 105 or 1 × 106 precultured cells. The differentiation ratio was statistically higher for implants that received 5 × 106 immediately implanted cells than for other implants at 7 days after implantation. At 14 days, implants that received 5 × 106 precultured chondrocytes had a higher differentiation ratio than did implants that received 5 × 106 chondrocytes that had not been precultured. Among implants that received precultured chondrocytes, total glycosaminoglycan and chondroitin sulfate content was lowest for implants that received only 1 × 105 cells. Among implants that received chondrocytes that had not been precultured, glycosaminoglycan content was not significantly different among the 3 cell concentrations, and chondroitin sulfate content was different only between implants that received 5 × 106 vs 1 × 106 cells. Only after the longest incubation period and at the highest cell concentration studied did preculturing of chondrocytes improve maintenance of phenotype. Preculturing did not appear to influence proteoglycan synthesis.

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