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. DESIGN: 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).

Hassall's corpuscle: a regressive formation.

Alpha naphthylesterase and leucineaminopeptidase activities in Hassall's corpuscles in the Gallus dom, and Cavia cobaya were found to be similar to those in man and in other animals. Histochemical findings of the thymus during the course of the growing phase have shown that these two enzymes are closely connected with the initial formation of a Hassall's corpuscle. Some medullary parenchymal epitheliocytes react strongly at this time. At first present in small clusters, they gradually become more numerous and finally escape from the network. Next they tend to cluster together, enclosing other cells with them. This change is connected with a new structure acquired by the cells. Enzymatic activity, antigenic properties and immunological reactions are only present in the early growth stages of a Hassall's corpuscle, when the cells are often hypertrophic. The component parts of the lamellated corpuscle are subject to involutive phenomena; they cease to function and to supply structural material until they finally disappear. The thymus is able to provide formative material without having recourse to Hassall's corpuscle. However, we are inclined to consider the progressive phase of Hassall's corpuscle to be important, as then elements in growth are clearly differentiated.