In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Abstract

Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CMR filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when beta-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.

Formation of Biphasic Constructs Containing Cartilage with a Calcified Zone Interface

“…Previous studies using deep-zone chondrocytes grown on filter inserts have indicated that the number of deep zone cells in culture could influence the amount of mineralization. 6 In that study, the cultures were established at the same cell density, but dilution of the deep-zone cells with chondrocytes from the superficial zone resulted in a decrease in size of the mineral deposits. During the harvest of cartilage in our study, we observed that the thickness of the deep and calcified regions of cartilage varied between cows, thereby influencing the number of deep-zone chondrocytes with the capability to initiate mineralization obtained from each cartilage harvest.…”

“…6,7,10 Cells from 2 to 3 animals were pooled to obtain sufficient cells for each experiment. The cells were placed on the upper surface of the porous CPP discs at a cell density of 160,000 cells/mm 2 .…”

“…5 This feature of the cartilage-substrate interface region has generally not been considered in the design of tissue-engineered osteochondral implants. The purpose of the present study was to develop a method for forming such an intermediary calcified zone within the in vitro-formed biphasic tissue-engineered constructs using techniques previously developed in our laboratory 6,7 and to characterize the resulting triphasic (i.e., cartilage-calcified cartilage-calcium polyphosphate (CPP)) structure. The bone substitute component of the constructs was made from CPP powders that were sintered to form porous cylinders with porosity suitable for anchoring in vitro-formed cartilage at one surface while retaining an open-pored structure throughout the remainder of the construct.…”

Engineering Complex Tissues

“…This approach allows generation of cartilage with a calcified zone. [120][121][122][123] By using chondrocytes isolated specifically from the deep zone of articular cartilage, it is possible to generate cartilage with a mineralized zone adjacent to the bone substitute (Fig. 10).…”

Zelluläre, molekularbiologische und morphologische Untersuchungen zur autologen Chondrozytentransplantation (ACT)

“…Weiters zeigen Chondrozyten aufgrund der zonalen Gliederung des Knorpelgewebes unterschiedliche biologische Eigenschaften, die auch in der Zellkultur nachweisbar sind [20]. Bei der ACT wird bei der Kultivierung der Zellen keine Ru Ècksicht auf die Subspezialisierung der Zellen genommen [4].…”