Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR.
OBJECTIVE
In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed.


METHODS
25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR).


RESULTS
The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors.


CONCLUSIONS
While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.
BG Berlin-Genetics GmbH, MDC-Buch, Berlin, Germany. stumm@kudamm-199.de
M
M
Stumm
E
E
Boger
C G
CG
Gaissmaier
C
C
Oßwald
M
M
Blankenburg
R D
RD
Wegner
J A
JA
Mollenhauer
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism*; D016022:Case-Control Studies; D019902:Chondrocytes / Q000378:metabolism*; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D055028:Comparative Genomic Hybridization; D005260:Female; D050173:Genes, Y-Linked; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D053583:Interleukin-1beta / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012737:Sex Factors; D055815:Young Adult
D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D053583:Interleukin-1beta; D012333:RNA, Messenger