Abstract
Tissue engineering techniques for cartilage re-pair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back into the patient＞s joint de-fect. The defect is then subsequently filled by new cartilage. Whether the cells in the repair tissue originate from the engineered tissue of the host or are derived from the surrounding original cartilage remains a relevant question for the ap-plied therapy. To answer this several methods exist to track cells, such as transfection of cells with LacZ carrying viruses, radio labeling with 111 IN or 51 Cr or fluorescent labeling with FDA. However, these techniques have drawbacks such as they may influence cellular properties, are radioactive and or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26劌 is a relatively new cell-labeling agent, but few data exist on the application of this dye in chondrocytes in vitro and in vivo. 5-chloromethylfluorescein diacetate - CMFDA (¨cell tracker green〔) is an established fluores-cent probe for imaging the dynamic processes of cell proliferation in vitro and in vivo. Likewise, several studies exist on different cell types. However, little data are available for chondro-cytes. The first aim of the study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influ-ence of labeling with CMFDA on cellular adhe-sion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as stan-dard and PKH 26潴 for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effec-tively with both PKH 26潴 and CMFDA. The PKH 26潴 labeled articular chondrocytes maintained fluorescence longer than CMFDA in vitro and in vivo. A higher percentage of articular chondro-cytes remained stained at 63 days than auricular or costal chondrocytes.
Highlights
Applied tissue-engineering techniques are clinical reality in joint cartilage replacement and defect filling
We show that chondrocytes from different origin can be labeled effectively with both PKH 26® and CMFDA
We evaluated the differences in PKH 26® and CMFDA durability of cell labeling in vitro for 9 weeks for 3 daughter cell generations and in vivo for 12 weeks in a long-term study
Summary
Applied tissue-engineering techniques are clinical reality in joint cartilage replacement and defect filling. The relevant question to justify the technique remains weather the defect surrounding chondrocytes are migrating into the lesion or if the new tissue originates from the implanted cells. To answer this durable and stable methods for cell staining are essential in long term follow up studies of transplanted cells in order to allow identification of the origin of the daughter cells. The marker should be 1: detectable after a prolonged period of time, 2: be easy to handle, and 3: should not interfere with immunocytochemical staining to characterize the fate and differentiation of grafted cells in the host tissue, such as cartilage
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