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Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients

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Abstract

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

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Abbreviations

ACTB:

Beta actin

AGC1:

Aggrecan 1

AMV:

Avian Myeloblastosis Virus

APA:

Alginate-polylysine-alginate

BGN:

Biglycan

CHES:

2-N-cyclohexylaminoethane sulfonic acid

COLIA1:

Type I alpha 1 collagen

COLIIA1:

Type II alpha 1 collagen

COLXA1:

Type X alpha 1 collagen

DCN:

Decorin

D-MEM:

Dulbecco’s modified Eagle’s medium

DT:

Doubling time

FCS:

Fetal calf serum

GAGs:

Glycosaminoglycans

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

OA:

Osteoarthritis

PAS:

Periodic acid-schiff

PBS:

Phosphate buffer saline

PCR:

Polymerase-chain reaction

RT:

Real time

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TEM:

Transmission electron microscopy

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Acknowledgments

This work was supported by a grant from the Fondazione Ente Cassa di Risparmio di Firenze to Maria Luisa Brandi.

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Authors and Affiliations

  1. Departments of Internal Medicine, University of Florence, Viale Pieraccini, 6, 50139 , Florence, Italy

    Anna Maria Carossino, Raffaella Recenti, Roberto Carossino, Elisabetta Piscitelli, Alessia Gozzini, Valentina Martineti, Gianna Galli, Isabella Tognarini, Alberto Moggi Pignone, Mario Cagnoni & Maria Luisa Brandi

  2. Departments of Human Pathology and Oncology, University of Florence, Florence, Italy

    Alessandro Franchi & Daniele Danielli

  3. Departments of Orthopedics, University of Florence, Florence, Italy

    Paolo Aglietti & Antonio Ciardullo

  4. DeGene Spin-off, University of Florence, Florence, Italy

    Valentina Martineti, Carmelo Mavilia & Maria Luisa Brandi

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  1. Anna Maria Carossino
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  2. Raffaella Recenti
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  3. Roberto Carossino
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  4. Elisabetta Piscitelli
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Correspondence to Maria Luisa Brandi.

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Carossino, A.M., Recenti, R., Carossino, R. et al. Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients. Biogerontology 8, 483–498 (2007). https://doi.org/10.1007/s10522-007-9088-4

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