MSC obtained from 4 different patients each were used throughout the individual differentiation pathways for this study. As was described previously 36 monolayer cultures treated with osteogenic supplements showed a high percentage of alkaline-positive cells, stained positive for mineralized matrix deposition and expressed the osteogenic specific marker genes alkaline phosphatase and osteocalcin (Fig. 1). Monolayer cell cultures treated with adipogenic supplements showed cytoplasmic lipid droplets and expressed LPL and PPARγ2, characteristic for adipocytes (Fig. 2). High density pellet cell cultures were composed of morpholocically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix and expressed collagen type II (Fig. 3).

Bone marrow-derived MSC prior to the initiation of the various differentiation pathways expressed CYR61/CCN1, CTGF/CCN2, CCN5 and CCN6 genes by RT-PCR analysis at high levels (Fig 4, 6 and 7, left lane each). No signals were obtained for CCN3 and for CCN4 expression in undifferentiated MSC as well as during differentiation pathways derived thereof, even after variations of the RT-PCR procedure. Further analyses, therefore, focused on CYR61/CCN1, CTGF/CCN2, WISP2/CCN5 and WISP3/CCN6 (see below).

A marked decrease in the expression of CYR61/CCN1 was observed during osteogenic differentiation. At the mRNA level RT-PCR analysis revealed a 6.9 ± 2.2-fold decrease (n = 4) in CYR61/CCN1 levels after 4 weeks of osteogenic differentiation, compared to undifferentiated MSC (Fig. 4). RT-PCR analysis for other CCN-members in this study did not display changes in PCR product intensity during osteogenic differentiation. At the protein level, immunocytochemistry showed a decrease in CYR61/CCN1 signal from differentiated cells compared to undifferentiated MSC (Fig. 5).

RT-PCR analysis of the CCN family members during adipogenic differentiation is shown in Fig 6. CYR61/CCN1 and WISP2/CCN5 were expressed prior to the initiation of adipogenesis and in initial stages of the differentiation pathway. However, in differentiated adipocytes after 2 weeks of differentiation almost undetectable levels were observed (n = 4). Due to the low expression levels densitometry was not possible. RT-PCR for CTGF/CCN2 revealed a slight reduction of the PCR product levels towards differentiated adipocytes (2.1 ± 0.8-fold). WISP3/CCN6 PCR product intensity did not change during adipogenic differentiation.

In high-density cell pellets prior to the initiation of chondrogenic differentiation RT-PCR analysis for CYR61/CCN1 and WISP3/CCN6 revealed high levels of RNA expression. During differentiation after one week signals for CYR61/CCN1dropped to undetectable levels. (Fig. 7). Therefore, densitometry for CYR61/CCN1 RT-PCR products after differentiation was not possible. PCR product intensity for WISP3/CCN6 was reduced 5.5 ± 0.6-fold (n = 4). RT-PCR for CTGF/CCN2 revealed a slight reduction during chondrogenic differentiation of 1.9 ± 0.6-fold. WISP2/CCN5 PCR products did not reveal differences in intensity along the chondrogenic differentiation of MSC. Immunohistochemistry of slides cut through the center of the pellets indicated high levels of CYR61/CCN1 protein prior to the initiation of differentiation. After three weeks of chondrogenic differentiation a reduced signal intensity at the periphery of the pellet was observed (Fig. 8).

FCS was supplied by Gibco (Eggenstein, Germany). Taq Polymerase was purchased from Amersham Pharmacia (Freiburg, Germany). An anti mouse CYR61 polyclonal antiserum was obtained from Munin Corp. (Chicago, USA). All other chemicals were of the highest purity available.

MSC were isolated from human bone marrow obtained from the femoral head of patients undergoing total hip arthroplasty using a modified protocol 36 originally described by Haynesworth et al. 31. Usage of patient material was approved by the local ethics commitee of the University of Würzburg and written consent was obtained from each patient. MSC from 4 patients were used for this study (3 male, 1 female), age between 51 and 62 years. Trabecular bone plugs were harvested from the cutting plane of the femoral head and transferred to 50 ml conical tubes containing DMEM/F12 medium (PAA, Cölbe, Germany). After vortexing and centrifugation (1000 rpm, 5 min) the pellet containing bone plugs and released cells was reconstituted in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), antibiotics (50 I.U. penicillin/ml and 50 μg streptomycin/ml) and 50 μg/ml ascorbate (complete medium). After repeated washings, the released cells were pelleted (1000 rpm, 5 min), suspended in complete medium, plated at a density of 60 × 10⁶ cells per 150 cm² tissue culture flask and maintained at 37°C in 5% CO₂. Non adherent cells were removed after 2 days and subsequently the medium was changed every 2 days until the cell cultures reached confluency.

Cells were plated in 6-well plates or chamber slides in complete medium. At confluency osteogenic differentiation was initiated using the complete medium supplemented with 10 mM β-glycerophosphate and 50 μg/ml ascorbate. Medium was changed every 2 days up to 4 weeks. To monitor osteogenic differentiation RT-PCR analysis for alkaline phosphatase, osteopontin and osteocalcin was performed as well as stainings for alkaline phosphatase (Sigma, Taufkirchen, Germany, Kit No. 86) and calcium phosphate deposition (Alizarin Red S) were performed as described by Nöth et al. 36.

Cells were plated in 6-well plates or chamber slides in DMEM with 10 % FBS. At confluency, adipogenic differentiation was initiated using the same medium supplemented with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 μg/ml insulin and 100 μM indomethacin. Medium was changed every 2 days up to 2 weeks. To monitor adipogenic differentiation RT-PCR analysis for lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ2 (PPARγ2) was performed, and a staining for lipid droplets (Oil Red O) as described 3336.

Cells were cultured as high-density pellet cultures in a serum-free medium as described previously 3233. 200, 000 cells were centrifuged for 5 min and 1000 rpm in 15 ml conical tubes and the pellets cultured for 3 weeks at 37°C in 5 % CO₂. The chondrogenic medium consisted of DMEM supplemented with 10 ng/ml transforming growth factor β1 (Stathmann, Hamburg, Germany), 100 nM dexamethasone, 50 μg/ml ascorbic 2-phosphate, 100 μg/ml sodium pyruvate, 40 μg/ml proline and ITS-plus (consisting of 6.25 μg/ml bovine insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.33 μg/ml linoleic acid and 1.25 mg/ml bovine serum albumin; Sigma, Taufkirchen, Germany). Medium was changed twice per week. To monitor chondrogenic differentiation RT-PCR analysis for aggrecan and collagen types I, II, IX and X was performed. Additionally, sections were cut through the center of the pellets and stained with alcian blue and were subjected to immunohistochemical analyses for collagen type II, chondroitin sulphate 4 and 6 as described by Nöth et al. 36.

RNA isolates were obtained from cells within the differentiation pathways as well as control isolates. Controls for monolayer differentiation (i.e. into adipocytes and osteoblasts) represent confluent resting MSC at day 0 prior to the initiation of cell differentiation. Control RNA isolates for the chondrogenic differentiation pathway were done from pellets prior to the initiation of chondrogenic differentiation at day 0. RNA isolation was performed using purification columns (Qiagen, Hilden, Germany) according to the manufacturer. Cells were rinsed with PBS, lysis buffer was added, RNA was column purified and the yield was determined by spectrophotometry. cDNA synthesis was done in a 20 μl reaction using Super Script II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) with identical amounts of RNA isolated from individual differentiation pathways of MSC (1 μg). PCR was performed using a PTC-200 thermal cycler (MJ Research) in a volume of 30 μl containing 1 μl cDNA with primers (Table 1) for CCN family members as well as the housekeeping gene actin. Primers used for cell markers are presented in Table 2. The PCR buffer consisted of 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.25 mM dNTP's, 5 pmol forward and reverse primer, and 0.5 units of Taq polymerase. Cycling was 4 min at 94°C, 25 – 40 cycles (25 cycles: actin and E1α, 30 – 37 cycles for CCN members) of 94°C for 30 s, 58 – 64°C for 1 min (depending on the primers) and 72°C for 1 min, with a final 72°C step of 5 min. Sequence analysis was performed by dye terminator sequencing of the PCR products to verify specificity using an ABI 310 Sequencer. 10 μl of PCR products were separated on a 2% agarose gel containing ethidium bromide. In cases of differential PCR product intensities densitometry was performed using an LTF densitometer and the bioprofile software (LTF, Wasserburg, Germany)

MSC undergoing osteogenic differentiation were subjected to immunohistochemical analysis for CYR61 expression. Cells were rinsed with PBS, covered with a glass cover slip and analyzed immediately. As the primary antibody an anti mouse CYR61 polyclonal antiserum (Munin, Chicago, USA) at a 1:100 dilution in PBS was used and the slides were incubated at 4°C overnight. The further steps were carried out at room temperature. Following three 5 min washes with TBS a monoclonal mouse anti rabbit IgG (DAKO, Hamburg, Germany) antibody at a 1:50 dilution in a solution consisting of 100 μl human AB plasma in 700 μl PBS was added and incubated for 30 min. After rinsing with PBS a rabbit anti mouse IgG antiserum (DAKO, Hamburg, Germany) diluted 1:25 was added and incubated for 10 min. After rinsing in PBS cells were incubated with a complex of intestinal alkaline phosphatase and mouse monoclonal anti alkaline phosphatase antiserum (APAAP) (DAKO, Hamburg, Germany) at a 1:50 dilution for 10 min. The final signal intensity was amplified using 2 additional cycles of incubations with the rabbit anti mouse IgG antiserum and the APAAP complex. Signals were developed using a conventional freshly prepared fast-red staining solution (40 mg fast red, 18 mg levamisole and 20 mg naphtol-AS-MX-phosphate, 1 ml DMF and 40 ml propanediol-buffer consisting of 50 mM 2-amino-2 methyl 1,3 propanediol pH = 8.7). Following a 10 min incubation, sections were washed in ddH₂O and prepared for microscopy.

During every analysis a negative control was performed according to Wong et al. 22. Sections were incubated using a rabbit serum instead of the primary antibody (rabbit anti CYR61 antiserum) at a similar protein concentration. Microscopic images were acquired using a digital camera and IP-Lab-Spectrum analysis software.

Immunohistochemical analyses for markers of chondrogenic differentiation were performed using the antisera. Immunoreactivity was detected using a streptavidin-peroxidase staining as described 36.