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Chondrocyte viability in osteoarticular allografts may be exaggerated
In award-winning paper, researchers question how effective these chondrocytes really are after implantation.
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HERSHEY, Pa. — More orthopedic surgeons are turning to osteoarticular allografts as a treatment for focal osteoarticular defects, but University of Iowa investigators report actual chondrocyte viability may be less than optimal.
The researchers also suggested that traditional clinical research methods used to assess chondrocyte viability may not accurately indicate whether cells are functioning in ultrastructural imaging and cell cultures.
In the Cabaud Award Presentation at the American Orthopaedic Society for Sports Medicine 2006 Annual Meeting, held here, Annunziato Amendola, MD, reported on the results of a controlled laboratory study conducted at the university involving eight allograft and four fresh amputation cartilage samples. Amendola and colleagues analyzed the samples using viability stains in parallel with alternative methods, including in situ ultrastructural analysis of chondrocyte morphology and in vitro growth of chondrocytes isolated from cartilage specimens. They tested specimens fresh as well as after freeze thawing.
Using Calcein AM/EtHD, a cell viability and cytotoxicity staining method, the researchers accurately reported cell viability in fresh cartilage, which was confirmed by positive culture results. There were also a nearly equivalent number of cells detected using DAPI staining.
The low degree of cellular viability shown in freeze-thawed fresh cartilage by Calcein AM/EtHD was also consistent with negative cell culture results, according to the abstract.
However, the stains produced misleading results when applied to commercial allograft cartilage. For these samples, the stains failed to detect the near complete loss of intact chondrocytes. The researchers confirmed the findings from negative cell culture results and low levels of DAPI stain detection.
Amendola also reported the results of ultrastructural imaging tests with electron microscopy. The researchers found that most cells in some allograft cartilage specimens appeared to be in various stages of degeneration.
As a whole, these findings suggest that Calcein AM/EtHD stains “seriously overestimated the number of viable cells in commercial allograft cartilage,” the researchers wrote.
“What we found from our data is that [orthopedic researchers] should not rely solely on Calcein AM/EtHD live/dead staining for determining chondrocyte viability in allografts,” Amendola said.
For more information:
- Amendola A, Martin J, Lightfoot A. Chondrocyte viability in osteoarticular allografts: Are we overestimating chondrocyte viability? Presented at the American Orthopaedic Society for Sports Medicine 2006 Annual Meeting. June 29-July 2, 2006. Hershey, Pa.