22. IN VITRO CALCIFICATION OF IMMATURE BOVINE ARTICULAR CARTILAGE AND GROWTH PLATE: FORMATION OF A FUNCTIONAL ZONE OF CALCIFIED CARTILAGE

Department: Bioengineering
Faculty Advisor(s): Robert Sah

Primary Student
Name: Jennifer Hwang
Email: jehwang@ucsd.edu
Phone: 858-534-5682
Grad Year: 2010

Abstract
Introduction: The zone of calcified cartilage (CC) anchors articular cartilage (AC) to subchondral bone through a layer of intermediate stiffness that reduces stress gradients at the osteochondral interface. Factors affecting cartilage calcification have been assessed with zonal subpopulations of articular chondrocytes, differing in propensity to calcify in vitro. Explant cultures could provide a useful model to delineate relationships between metabolism and mechanical function for cartilage calcification. The objective of this study was to assess zonal variations in structure, indentation stiffness, and calcium uptake after in vitro culture of immature AC, as well as growth plate cartilage (GPC), under conditions promoting calcification. Methods: Cartilage was harvested from the patellofemoral groove of 5 immature bovine knees for AC and from between the epiphyseal and metaphyseal bone of 3 fetal bovine distal ulnae for GPC. Cartilage was cut into strips preserving zonal structure and incubated for 0-3 wk in DMEM, 1% FBS, 100 μg/mL ascorbic acid, ±10 mM ß-glycerophosphate (ßGP), and ±0.5 μCi/mL 45Ca++. The time course of calcification was assessed by comparing groups analyzed fresh and after 1, 2, or 3 weeks of culture with +10mM ßGP. The biological mechanisms of calcification were analyzed, including the role of viable cells and the role of medium supplementation with ßGP. After termination, cultures were analyzed by histology for structure, by indentation for stiffness, and by 45Ca++ uptake for calcium accumulation. Results: The extent of calcification was dependent on the tissue zone and the culture time for both AC and GPC. By week 3 of culture (+10mM ßGP), the deep ~25% of AC and metaphyseal ~60% of GPC strips not only appeared brown macroscopically, but was stained with Alizarin Red S in tissue around cells in AC and between cell columns in GPC. At week 3, the deep 20% of AC was 3-fold stiffer than the region 0-60% from the articular surface (p<0.05). Calcification was dependent on live cells, and, for AC, ßGP supplementation of medium. Conclusions: Zonal variations in structure, stiffness, and Ca++ uptake indicate that the deep 20% of immature AC can undergo selective physiological calcification within 2-3 weeks of explant culture. The controlled formation of a CC-like tissue would facilitate the biomimetic attachment of a cartilaginous tissue to a bony substrate to form a mechanically functional osteochondral unit.

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