Materials and Methods

Chemicals and Supplies

• Abstract
• Results
• Discussion
• References

Bovine (b) and human (h) PTH fragments were obtained from Sigma (St. Louis, MO): h, b(PTH) bPTH (1-34), (Nie 8,18,Tyr 34) bPTH (3-34), hPTH (13-34), hPTH (28-48), hPTH (39-68),
(Tyr52, Asn 76) hPTH (52-84), and hPTH (64-84).

Tissue culture supplies were purchased from Becton Dickinson (NJ), and PCS from PAA-Labor, Forschungs GmbH (Linz, Austria).

Northern Blot
Chondrocytes were lysed in 4 M guanidinium thiocyanate, 25 mM Nacitrate, 0,5% Na-sarcosyl, and 0.7% ß-mercaptoethanol. RNA was extracted using the cesium-chloride density centrifugation method of Chirgwin et al. (9). Total RNA (10 u-g) was subjected to formaldehyde
gel electrophore-sis, blotted onto nylon filters and cross-linked by exposure to UV light for 5 min. For analysis of RNA, cDNA probes were labeled with [32P]dATP or dCTP by random priming and hybridized in 50% formamide, 5x SSC, 5x Dehnhardt’s solution (4), 0.5% SDS, and 100
(ig/ml herring sperm DNA at 42°C for 16 h. After hybridization, filters were washed twice in 2X SSC at room temperature for 5 min, twice in 2x SSC/0.1% SDS each at 50°C for 30 min, and once in 0.1 X SSC/0.1% SDS at room temperature for 2 min.

The washed filters were exposed to Kodak X-OMAT™ X-ray films (Eastman Kodak).
Microfluorometry of Ca2+-Signaling For Ca2+ imaging, chondrocytes were seeded on 35-mm tissue culture dishes and incubated in Harn F-12 for 24 h before loading with Fura-2/AM (5 u,M) for 30 min at 37°C. Ca2+ was imaged with an upright microscope (Zeiss Axioskop FS, Jena, Germany) and a 40X water Immersion objec-tive. A CCD camera System (Photometrics Ltd., Tuscon AZ) (12, 37, 38) was used to acquire digitized Images of Fura-2 fluorescence. Free Ca2+ concentrations were determined from background corrected image pairs at 350 and 380 nm excitation with the ratio method (18).

The responsive-ness of the calcium signaling machinery of the chondrocyte population was controlled by the Ca2+ response to 5 u,l FCS.
Subsequently, the return of intracellular free Ca2+ to stable baseline levels was recorded for at least 10 min before the application of PTH fragments.
Cells were continuously superfused with saline containing NaCl 140 mM, KC15 mM, MgSO4 2 mM, NaH2PO4 l mM, glucose 5.5 mM, Hepes 20 mM, pH 7.4. The PTH pep-tides were applied by microdrop application of concentrated stock solution into the bath to give final concentrations äs
indicated. When different PTH fragments were sequentially tested on the same chondrocyte population, a return to baseline Ca2+ fluorescence and stability for 10 min was imperative before application of a new peptide.

Agarose Cell Culture
Juvenile human costal cartilage obtained from funnel chest operations was dissected free of surrounding tissues and cut into 0.5-mm slices. Chondrocytes were released by collagenase digestion and cultured in agarose gels under serum-free conditions as described previously (6, 53). Briefly, matrix-free cells suspended in media containing 0.5% of low melting agarose
were seeded into prewarmed culture dishes coated with 1% high melting agarose gels. The cultures were maintained at 37°C to keep the low melting agarose in the liquid state and, thus, to allow the cells to sediment at the interface of the two agarose layers. Thereafter, the low
melting agarose was allowed to gel by brief exposure of the cultures to 4°C. Cultures were then supplemented with additional medium.

Cells were grown at densities of 2 X 106/ml in DMEM (Grand Island Biologicals Corp., Basel, Switzerland) containing 60 mg/ml of ßaminoproprionitrile fumarate, 50 mg/ml sodium ascorbate, l mM cysteine, l mM pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin. Where applicable, PTH fragments were added for 24 h. Subsequently, the media were exchanged and the cultures were maintained for another 48 h in the presence of l mCi [14C]proline (uniformly labeled,285 mCi/mmol, Amersham International) and analyzed for collagen synthesis.

The radiolabeled agarose cultures were homogenized and newly synthesized collagens extracted by digestion with pepsin (6). The total extracted proteins were precipitated with ethanol and analyzed by SDSPAGE (4.5-15% gradient gel) followed by fluorography.

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