Human chondrocyte-conditioned medium promotes chondrogenesis of bone marrow stem cells

Human chondrocytes were isolated from redundant cartilage of 43–75 years old consented patients from Southeast Asia; undergoing total knee replacement surgery. Only the normal cartilage samples proximal to the affected area were processed within 24 h of surgery. The cartilage was finely minced and immediately digested with 0.6% Collagenase type II for 3 to 4 h, at 37ºC under continuous agitation. After that, the suspension of digested cartilage was centrifuged I and the pellet containing isolated cells were seeded with Chondrocyte Medium (PromoCell, USA). For BMSCs, aspirated bone marrow was diluted at a 1:1 ratio with F12:DMEM (FD) medium supplement with 10% foetal bovine serum (FBS). The diluted bone marrow was layered over 10 ml of Ficoll-Paque, and centrifuged at 2,500 rpm for 30 min. Mononuclear cells were collected at the interface, and then suspended in PBS before being centrifuged at 5,000 rpm for 15 min. The pellet was then resuspended in FD medium supplemented with 10% FBS and was seeded in a 6-well plate. Once both cells were confluent, they were sub-cultured at 5,000 cells/cm² until passage 3. All culture media were changed on alternate days.

Confluent chondrocyte culture was used to prepare CCM. Cells were washed with Dulbecco Phosphate Buffer Saline (DPBS) twice and then supplemented with FD without FBS. The CCM was collected after 48 and 72 h of culture. This step was repeated for passage 1 to 3. CCM was stored at −20°C until use. The ratio of conditioned medium that yielded the highest chondrogenic differentiation was examined by culturing the MSCs for 7, 14, and 21 days in FD medium containing 25% and 50% (v/v) conditioned medium.

Culture supernatants were collected from each sample and protein quantification was performed using the BCA assay kit (SIGMA). The absorbance was read at 562 nm using a spectrophotometer (BioTek PowerWave XS, Biotek, USA). The standard curve was plotted using BSA protein standards from 0 to 1000 mg/ml and the amount of protein in CCM was calculated.

BMSCs were induced using CCM and chondrogenic induction medium (CIM). Non-induced BMSCs were used as a control. For the induction, 25% and 50% (v/v) CCM in FD medium were supplemented to BMSCs. Chondrogenic Induction Medium (CIM) was prepared as described previously [1]. Confluent BMSCs at passage 3 were induced with CIM or CCM for 21 days and the media were changed every 3 days. The induced BMSCs with CIM and CCM are hereafter referred to as iCIM and iCCM, respectively.

Cells were washed with DPBS prior to fixing with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Then, the cells were permeabilised for 20 min with 0.5% Triton X-100 solution (Sigma-Aldrich), and blocked with 10% goat serum (Sigma-Aldrich) for 1 h at 37°C. The cells were subsequently incubated with 1:200 rabbit anti-collagen 1 (TA309096; OriGene, USA) and 1:200 mouse anti-collagen II antibodies (ab185430; Abcam, USA) overnight at 4°C. On the following day, the cells were washed before being incubated with Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, USA) and Alexa Fluor 488 goat anti-mouse (Invitrogen, USA), respectively, for 1 h at 37°C. Then, nuclei were counterstained with 1:20,000 4’,6-diamidino-2-phenylindole (DAPI) before being observed using a Nikon Eclipse Ti fluorescence microscope (Nikon, Japan). Immunofluorescence staining was performed on 7, 14, and 21 days post-induction of BMSCs.

Cells were washed with DPBS prior to fixation with 10% formalin. After 10 min, the fixative was removed, and each well was washed twice with DPBS. Then, fast green solution (Dako, Denmark) was added to the well for 5 min before rinsing with 1% acetic acid for 15 sec. Then, 0.1% Safranin-O (Sigma-Aldrich) solution was added to the well for 5 min. Finally, the cells were washed twice with DPBS and observed under Olympus CK40 inverted microscope (Olympus, Germany).

Cells were washed with DPBS prior to fixation with 10% formalin. After 10 min, the fixative was removed, and each well was washed twice with DPBS. Next, 0.04% toluidine blue (Gainland UK) was added to the well for 1 h at room temperature. Lastly, the cells were washed twice with DPBS and observed under Olympus CK40 inverted microscope (Olympus, Germany).

After 21 days of culture and induction, the expression of collagen type I (COL1), collagen type II (COL2), collagen type X (COLX), aggrecan core protein (ACP), and sex-determining region Y box 9 (SOX9) genes in chondrocytes (control), BMSCs (control), iCCM and iCIM was evaluated using real-time RT-PCR. The cell was trypsinized and centrifuged at 5000 rpm for 5 minutes at 37°C. The cells pellet was suspended and total RNA was extracted using column method by Qiagen RNeasy Mini kit (Qiagen, Germany) and quantified with spectrophotometer (BioTek PowerWave XS, Biotek, USA). RNA integrity was ensure by performing agarose gel electrophoresis together with Olerup SSP GelRed (CareDx, USA). A 20-μl aliquot of first-strand cDNA was synthesizes using the QuantiNova™ Reverse Transcription kit (Qiagen, Germany) according to the manufacturer's protocol. QuantiNova® SYBR® Green PCR (Qiagen, Germany) was used for quantitative polymerase chain reaction (qPCR) analysis using 2 μl of each reverse-transcribed sample. The assay was run in a StepOne Plus Real-Time thermal cycler (Applied Biosystems, USA) with the following PCR cycling conditions: initial denaturation for 2 min at 95°C, followed by 40 cycles of denaturation for 5 sec at 95°C, then anne-aling/extension for 10 min at 60°C for all the primer pairs. The assay was followed by a melt curve analysis to ensure PCR product specificity. Table 1 shows the primer sequence used for RT-PCR following previous studies [42, 43, 44]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The data were analyzed using The StepOne™ Software. For gene expression quantitation, the comparative Ct method was used. The Ct values of the gene of interest were normalized to GAPDH. The Ct value was calculated when the fluorescence of the sample exceeded threshold level.

Data are shown as mean of 3 biological samples ± SEM (n = 3). The comparison of the mean between groups was assessed with one-way ANOVA test of variance. P ≤ 0.05 was considered significantly different.

Protein concentration was found to be significantly higher in passage 3 CCM (1023.22 ± 55.85 μg/ml and 1108.64 ± 7.82 μg/ml for 48 h and 72 h incubation, respectively) compared to the CCM from other passages (Figure 1). There is no significant difference between the amounts of protein for CCM collected 48 h and 72 h post-culture in all passages. Therefore, medium that has been conditioned for 72 h using passage 3 chondrocytes was chosen for further evaluation.

Figure 1

As shown in Figure 2, BMSCs exhibited fibroblastic shape, while chondrocytes have more rounded and polygonal shapes with small extensions. Both type of cells grew in monolayers when cultured in their respective culture medium (FD+10% FBS and Chondrocyte medium). The iCIM started to form aggregates or clumps as early as day 7. Meanwhile, iCCM started to grow compactly in several spots; however, there was no prominent clump formation observed.

Figure 2

Type 1 collagen was expressed prominently in every time point for all groups. Meanwhile, Type 2 collagen was not expressed in the BMSC group throughout the culture period (Figure 3). Chondrocytes expressed collagen type 2; however, the expression became weaker with increasing culture time. For the iCIM and iCCM group, the treatment caused the induced BMSCs to express collagen type 2 throughout the culture period.

Figure 3

iCCM was stained with Safranin-O to test for their GAG synthesis ability. Non-induced BMSCs showed negative staining for Safranin-O while BMSCs induced with CCM and CIM showed the positive staining of Safranin-O as early as day 7 (Figure 4).

Figure 4

BMSCs showed no positive staining for toluidine blue. The iCIM and iCCM 50% on day 21 showed the prominent staining of toluidine blue similar to chondrocytes which showed the formation of proteoglycans and glycosaminoglycans by the induced cell (Figure 5).

Figure 5

The gene expression for type I, II, and X collagens, Sox9, and aggrecan core protein (a proteoglycan composed of GAGs) on day 21 was evaluated. As shown in Table 2, chondrocytes had the higher expression of collagen type 2 compared to BMSCs, iCIM and iCCM. There was no significant difference between the iCIM and iCCM group with regard to collagen type 2 expression; however, both groups expressed higher levels of collagen type 2 when compared to BMSCs. ACP expression was significantly higher in chondrocytes compared to BMSCs, but there were no significant differences in ACP expression between chondrocytes, iCIM and iCCM. Expression of the SOX9 gene in chondrocytes, iCIM and iCCM was higher compared to BMSCs. Besides that, iCIM and iCCM have significantly higher SOX9 expression than chondrocytes. The relative expression of collagen type 1 was the highest in BMSCs. CCM caused a reduction of collagen type 1 relative expression compared to non-induced BMSCs. For collagen type 10, BMSCs, iCIM and iCCM had lower expression levels compared to chondrocytes. The expression of collagen type 10 in iCCM was significantly higher compared to in iCIM and BMSCs.