Autologous chondrocyte implantation (ACI) for aged patients: development of the proper cell expansion conditions for possible therapeutic applications.
OBJECTIVE
Proliferation and chondrogenic commitment of cultured articular chondrocytes are impaired when cells derive from aged donors. In those subjects the feasibility of cell-based therapies for articular surface repair is reduced. Moreover, the use of serum as medium supplement elicits non-physiological responses in cultured chondrocytes. This study was therefore undertaken to identify the expansion culture conditions needed to sustain growth and chondrogenic commitment of chondrocytes harvested from aged human subjects.


METHODS
Articular cartilage was obtained from aged (69-75 years) and from young adult subjects (27-35 years). Chondrocytes were isolated and cultured in serum-free (SF) or in serum-supplemented [fetal calf serum (FCS)] conditions. Chondrocytes were expanded in monolayer for five duplications and processed for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The differentiation potential was assessed by micromass pellet cultures before and after expansion in either culture medium, or after a prolonged exposure to serum followed by a period in SF condition.


RESULTS
Only SF-cultured chondrocytes reached five duplications within 25-35 days, maintaining the expression of some chondrogenic markers and without altering the levels of active matrix metalloproteinase 3 (MMP-3). Only the pellets derived from SF-expanded cultures positively stained for cartilage matrix deposition. On the contrary, exposure to serum diminished the proliferation capacities, abolished the differentiation potential in the same cells and elicited transcription of the MMP-3 gene. Shifting culture conditions from FCS to SF resumed growth rates but proper extracellular matrix deposition was only partially restored.


CONCLUSIONS
The SF conditions have proven valuable to prime cell proliferation and to sustain proper commitment in chondrocytes from aged patients. This culturing approach may represent a therapeutic chance extendable to a range of patients normally excluded from clinical protocols based on autologous chondrocyte implantation (ACI).
Giannoni
P
P
Biorigen S.r.l., Via Peschiera 16, 16122-Genova, Italy. paolo.giannoni@istge.it
Pagano
A
A
Maggi
E
E
Arbicò
R
R
Randazzo
N
N
Grandizio
M
M
Cancedda
R
R
Dozin
B
B
D000328:Adult; D000368:Aged; D002358:Cartilage, Articular / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D016922:Cellular Senescence; D019902:Chondrocytes / Q000166:cytology / Q000637:transplantation*; D016895:Culture Media, Serum-Free; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods
D016895:Culture Media, Serum-Free