Greta M. Lee

ATP release by mechanically loaded porcine chondrons in pellet culture

OBJECTIVE To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

Isolated chondrons: a viable alternative for studies of chondrocyte metabolism in vitro

OBJECTIVE To develop and test a simple enzymatic procedure for isolating chondrons, which consist of the chondrocytes and their surrounding pericellular microenvironment. DESIGN Chondrons were obtained by digesting adult human articular cartilage with a mixture of dispase and collagenase. Chondrons and chondrocytes were cultured in alginate beads, immunofluorescence labeled and examined by confocal microscopy. RESULTS Comparison of freshly isolated chondrons with cryostat sections of cartilage revealed that type VI collagen, type II collagen and aggrecan were retained, but fibronectin and a unique chondroitin sulfate epitope recognized by the antibody, 7D4, were lost. Comparison of enzymatic and mechanical homogenization methods revealed subtle changes in chondron morphology and retention of fibronectin in mechanically isolated chondrons. Average yield of enzyme-isolated chondrons was slightly lower than that of chondrocytes isolated by pronase and collagenase digestion, but was much greater than that reported for mechanically isolated chondrons. Enzyme-isolated chondron viability was greater than 80% 1 day after isolation, and continued to be above 80% through 7 weeks of alginate bead culture. Viability of isolated chondrocytes was initially greater than 80% but fell to 60-80% with time in culture. Chondrons and isolated chondrocytes had a similar division rate except osteoarthritic chondrons were significantly slower after 2 weeks in culture. Cell division was more rapid for nonosteoarthritic chondrons than for osteoarthritic ones. CONCLUSIONS Enzymatic isolation of chondrons is relatively simple, gives better yield and viability than mechanical isolation, but comparable yield and viability of traditional chondrocyte isolation. Enzymatic chondron isolation allows the effect of the in vivo-formed pericellular matrix on chondrocyte metabolism to be studied in vitro.

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady‐state levels of phosphotyrosine‐modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF‐primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/trk tyrosine kinase because the presence of NGF in cultures of NGF‐primed cells was not necessary for genistein‐stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF‐primed PC12 cells. Video‐enhanced differential interference contrast microscopy revealed that growth cones of genistein‐treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage.

Tissue transglutaminase (tTG) catalyzes a Ca2+‐dependent transglutaminase (TGase) activity which cross‐links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg‐GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM, but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.

Extracellular nucleotides, cartilage stress, and calcium crystal formation.

&NA; Nucleotides are released by chondrocytes at rest and in response to mechanical stimulation. Extracellular nucleotides are metabolized by a variety of ectoenzymes, producing free phosphate (Pi) or pyrophosphate (PPi) and promoting matrix mineralization. Ectoenzymes are differentially localized in cartilage and may be co‐released with nucleotides during mechanical stimulation. Extracellular nucleotides can also serve as substrates and/or modulators of enzymes such as tissue transglutaminase and ecto‐protein kinases that modify matrix proteins and regulate crystal deposition or growth. Understanding the evolution of osteoarthritis and calcium crystal deposition diseases will require clearer knowledge of the functions of nucleotides and ectoenzymes in the cartilage extracellular matrix.

Development of selective tolerance to interleukin-1β by human chondrocytes in vitro

Interleukin‐1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL‐1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL‐1β. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL‐1β (0.05–10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL‐1β declined over the pretreatment period. In response to IL‐1β (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL‐1β on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL‐1β‐induced MMP‐13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL‐1β increased glucose consumption and lactate production throughout the treatment. IL‐1β stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non‐OA cartilage released the same amount of NO but produced less PGE2 and MMP‐13 in response to IL‐1β than OA cultures. Like the OA, IL‐1β‐induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL‐1β, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP‐13 production, metabolic activity, or matrix metabolism.

Response of Chondrocytes to Local Mechanical Injury in an Ex Vivo Model

Background: Our goal was to set up an ex vivo culture system to assess whether cartilage wounding (partial-thickness defects) can induce morphological changes in neighboring chondrocytes and whether these cells can translocate to the surface of the defect. Methods: Two-millimeter partial-depth defects were created in human osteochondral explants followed by culture for up to 4 weeks. Frozen sections of defects and defect-free regions were labeled using immunofluorescence for a plasma membrane protein, CD44, and actin with TRITC-phalloidin. Viable nuclei were detected with Hoechst 33342. Differential interference contrast (DIC), confocal, and transmission electron microscopy (TEM) were used to examine process extension. Results: Significant changes in cell morphology occurred in response to wounding in the superficial and deep cartilage zones. These included cell flattening, polarization of the actin cytoskeleton, extension of pseudopods projecting towards the edge of the defect, and interactions of these filopodia with collagen fibers. Cell density decreased progressively in the 300-µm zone adjacent to the defect to an average of approximately 25% to 35% after 3 weeks. Concomitant increases in cell density in the defect margin were observed. By contrast, minimal changes were seen in the middle cartilage zone. Conclusions: These novel observations strongly suggest active cartilage cell responses and movements in response to wounding. It is proposed that cartilage cells use contact guidance on fibrillated collagen to move into and populate defect areas in the superficial and deep zones.