To develop a physiological model for osteochondral tissue which allows for quick handling and high-throughput applications with limited human specimen at hand, we developed a method for the manufacturing of native osteochondral live slice cultures from human joint tissue. 500–800 µm thick osteochondral slice cultures were prepared from 23 different surgically explanted tibial plateaus with a special microtome insert and then cultivated in hanging inserts of 24-well plates (Fig. 1). In this study, we tested if the tissue cultures keep their physiological properties such as long-term cell viability, gene expression and responsiveness towards biological stimuli. In order to do so, tissue cultures of 6 donors were cultivated 7 days with additional stimulating cytokines and tissue cultures of 17 other donors were cultivated 21 days without any additional stimuli. Subsequently, the tissue cultures were analyzed microscopically, genetically and histologically.
Laser Microscopy shows highly conserved spatial cell order
In order to assess the viability and organization of cells in deep layers of cartilage tissue, culture slices were stained with PI/FDA and subsequently analyzed by CLSM. The amount of living cells was then compared to the results of the resazurin assay. Three-dimensional CLSM revealed < 10% chondrocyte viability in long-term heated slices, about 50% viability in short term heated slices and > 90% viability in non-heated slices (Fig. 2. A) after three weeks of prior cultivation time. Chondrocytes remained localized in their cartilage-typical, spatial alignment (column-like deep zone; pearl-bead structure in the mid zone; tightly packed cells in the superficial zone). No cell culture effects due to nutrition gradients were visible (e.g. no elongated cells in the peripheral tissue).
Resazurin assay correlates with visual assessed viability
To ensure that resazurin assay is sufficiently reflecting viability which might potentially be hampered by the dense ECM of osteochondral samples, selected samples of varying viability were analyzed with resazurin metabolic assay and CLSM. The results correlated with visually counted cells. A two-tailed Pearson correlation test showed a correlation ratio of R² = 80-84.5% of the metabolic assay with microscopical viability analysis (Fig. 2.C) which we regarded as sufficiently accurate to assess the general viability state of the tissue culture. Analysis of further tissue cultures was therefore performed with resazurin, since it allows for progredient viability analysis without the need of sacrificing the tissue slice with each measurement due to the toxic PI and hot laser beam of the CLSM.
Cell viability stays < 95% without additional serum over 21 days
After 7 days, F- cultivated slices of the cytokine stimulated groups exhibited a mean viability of 103 ± 28.3% compared to day 0 with a mean standard deviation between the stimulation groups of ± 6%. There was no significant difference between viability of day 0 and day 7 as well as between the four different cytokine stimulation groups. F + slices showed a significant increase in viability compared to day 0. Viability increased to a mean of 220 ± 60% with a mean standard deviation between the stimulation groups of 16.8% (Fig. 2. B). During the long-term cultivation, F- group showed viability of 114.24 ± 43.35% after 7 days, 108.1 ± 52.26% after 14 days and 113.37 ± 55.61% after 21 days. Statistical analysis showed no significant differences in viability between the time points (Fig. 2. B).
Figure 2 – Cell viability analysis. A. cartilage viability analysis using CLSM. Pictures show three different slices from the same donor with Propidiumiodid(red)/Fluoresceindiacetat(green) staining. To induce cell death for correlation analysis, (from left to right) the first slice was heated 1 hours at 60 °C, the second slice was heated 30 minutes at 60 °C and the third slice was left in its native state. Dead cells appear red, vital cells appear green. B. Metabolic viability analysis. Left chart shows mean viability on day seven of four groups stimulated with TNF-α (low 0.8 pM, high 1.2 pM) or TGF-β3 (800 pM) after seven days with(ν)- or without FBS (♦)-in relation to day (n = 6). Right chart depicts the cell viability in long-term cultivation after 7, 14 and 21 days; towards day 0, there was no statistically significant change detectable (n = 15). C. Correlation of visual and metabolic viability analysis. 13 slices of 2 different donors were analyzed microscopically and metabolically. For the individual donors, the two methods have a correlation value between 0.8 and 0.845.
ECM protein transcription stays highly reactive to external stimulation in serum-free cultivation
In order to test cell reactivity to external stimuli, we intended to induce visible changes in chondrocyte gene expression. Hence, slices were stimulated with TNF-α and TGF-β3 to either suppress or induce cartilage relevant gene markers. Gene expression analysis showed different results in F- and F + stimulation group. In the F- group, TNF-α significantly reduced expression of COL2A1, ACAN and COMP. In relation to day 7 control, COL2A1 expression decreased to 11 ± 7% at low dose TNF-α concentration and 5 ± 5% at high-dose TNF-α. ACAN expression decreased to 42 ± 33% in the lower concentration and 15 ± 12% in the high concentration. COMP expression dropped to 44 ± 48% in the lower concentration and to 10 ± 14% in the high concentration. TGF-β3 increased COMP expression 9-fold ± 5and COL1A1 expression by a mean foldchange of 326 - although with no statistical significance (± 434; p = 0.15) (Fig. 3.A.). In the F + group, TNF-α decreased COL2A1 expression to only 33 ± 35% in the lower concentration (p = 0.09) and to 11 ± 16% in the high concentration towards day 7 negative control. Mean expression levels also decreased in ACAN and COMP expression after TNF-α exposure, but the values exhibited lower significance than in the F- group (ACAN: p = 0.15 and p = 0.32; COMP 0.08 and 0.20). TGF-β3 significantly increased expression of COMP 29-fold (± 2.51; p = 0.005) and mean COL1A1 expression to 6-fold again with low statistical significance (± 6.84; p = 0.26) (Fig. 3.B.).
Effects on expression of proliferative markers are only distinguishable in serum-free cultivation
Expression analysis of proliferation marker Ki-67 in F- cultivated groups upon TGF-β3 stimulation showed an increase in expression levels after 7 days towards day 0 control by 3738 ± 5987%, though not statistically significant (p = 0.06). In the F + group, however, the negative control of day 7 and all 3 stimulation groups exhibited statistically significant changes towards day 0. Mean increase of expression of all 4 groups resulted in a 5.19 × 105 ±5.9 × 105 fold change (p = 0.003). No statistically significant differences were detected between the different F + cultivated stimulation groups in relation to day 7 negative control (Fig. 3. A. and B.).
Expression of essential cartilage ECM ACAN and COL2A1 proteins stays stable for about 14 days in unstimulated slice culture
Expression of cartilage relevant genes COL2A1 and ACAN stays stable for 14 days and then starts to significantly decline on day 21 (COL2A1: 32 ± 23%; and ACAN 51 ± 64% of day 0) (Fig. 3.C.). Cartilage remodeling marker COL1A1 shows a short increase after 7 days but then returns to levels of day 0 at day 21. Expression of COMP reacted very promptly to the cultivating situation with a significant drop in expression levels to 45 ± 30% after 7 days to 14 ± 11% after 21 days, respectively. Expression of Ki-67 as a marker for cell proliferation increases up to 100.000% overtime in relation to day 0 but the increase is inignificant and the high relative increase at the measurement points are a result of the very low absolute initial expression of Ki-67 at day 0.
Figure 3 – Gene expression analysis A. RT-PCR of stimulated F- cultivated slices. Bar charts show the quantitative gene expression of osteochondral slices stimulated with either low dose TNF-α (light grey), high dose TNF- α (dark grey) or TGF-β3(black) stimulated osteochondral slices in F- medium. Shown are cartilage relevant markers gene COL2A1, COL1A1, ACAN, COMP and the proliferation relevant gene Ki-67 after 7 days of cultivation in comparison to the negative control with Succinate dehydrogenase complex subunit A as reference gene. Scales are logarithmic for clear presentability (n = 6 donors). B. RT-PCR of stimulated F + cultivated slices. Graphs show the same setup as the graphs in A but with 10% FBS addition (n = 6). C. RT-PCR of long-term cultivated slice cultures. Gene expression of 21 days cultivated slices at day 7, 14 and 21 in relation to day 0 (n = 12 donors).
Tissue slices react to TNF- α with visible remodeling of ECM and cellular swelling
To analyze, whether the native slice cultures preserve their tissue-specific functionality to remodel extracellular matrix, slices were stained with Safranin-O. Proteoglycan content of TNF-α stimulated slices and negative controls was quantified via histomorphometry (Fig. 4. A). In accordance to the alterations in gene expression, TNF-α stimulated slices showed strongly altered histomorphology. Intra-cartilage fibers appeared less dense and chondrocyte increased in volume and diameter (Fig. 4. B). Proteoglycan content in the TNF-α-treated group was also significantly reduced: Intensity of Safranin-O staining declined by 32% ± 24 (p = 0.04) compared to the untreated controls (Fig. 4. C).
Figure 4 – Histological analysis (n = 6 donors) A. Histomorphometrical analysis – Histomorphometrical analysis of Safranin-O stainings of all experimental groups considering the proportion and intensity of the stained area as the mean intensity normalized to the control + standard deviation. B. Representative picture of TNF-treated slice and control. TNF-α-treated slices show less intense Safranin-O staining, loosened matrix structure and swollen cells in the mid zone of the cartilage. C. Comparison of color intensity in stimulated and non-stimulated slices. TNF-α stimulated slices show a mean reduction of red-intensity of 32% (n = 6).