Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

Human mesenchymal stromal cell (hMSC) isolation

Bone marrow was aspirated from patients undergoing total knee/hip arthroplasty in the University of Malaya Medical Centre. The study was approved by the Medical Ethics Committee of the institution (UMMC, reference number 967.10). Written informed consent was obtained from each participant. Aspirated bone marrow was added to equal volume of phosphate-buffered saline (PBS; pH 7.2; Invitrogen-Gibco, MA, USA) and layered onto Ficoll-Paque Premium of density 1.073 g/mL (GE Healthcare, Uppsala, Sweden). The samples were then centrifuged at 360× g for 25 min. The mononuclear cells were later isolated and resuspended in 10 mL of low glucose Dulbecco’s modified eagle medium (L-DMEM) (Invitrogen-Gibco) and centrifuged again at 220× g for 5 min. The supernatant was discarded and the cell pellet obtained was cultured in growth medium (L-DMEM supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (100 U/mL; Invitrogen-Gibco) and 1% Glutamax-1 (Invitrogen-Gibco) in T-25 tissue culture flasks. Medium was changed every three days until the cultures were 80% confluent. The cells were then serially passaged until passage 2, which were harvested and used for further experiments.

Before proceeding with the experiment, we performed additional experimental procedures to verify that the isolated cells were mesenchymal stromal cells, based on their ability to differentiate to adipocyte, osteoblast and chondrocyte. The experiments were carried out as described earlier (Nam et al., 2013). The formation of lipid droplets and mineralization indicated the potential of cells to differentiate to the adipogenic and osteogenic lineage, respectively. The ability of the cells to differentiate to chondrocytes was reflected by the intensity of the red staining for proteoglycans deposited in the matrix. Our results confirmed that the isolated cells were mesenchymal stromal cells (data not shown).

Preparation and addition of PRC

PRC was prepared from a pool of blood from six healthy volunteers. Blood samples for PRC preparation and bone marrow harvested for hMSCs isolation originated from different group of donors. Twenty-five milliliters of blood from each subject was collected in ACD tubes. Platelets were prepared using double centrifugation method as mentioned previously, with slight modifications (Cho et al., 2011; Lee et al., 2012; Shani et al., 2014). In brief, the first centrifugation was at 200× g for 10 min, which separated the red blood cells, plasma and buffy coat that contains the platelets and the white blood cells. The plasma and buffy coat obtained were pooled in a 50 mL falcon tube in order to minimize inter-individual variability. Prostacyclin (PGI₂) was added to prevent the transitory activation of platelets during the centrifugation and re-suspension steps. The pooled plasma was then centrifuged at 800× g for another 10 min. The supernatant portion of the plasma was discarded and only the platelet pellets were isolated and resuspended in sterile phosphate buffered saline (PBS, pH 7.2), at 1/10th the initial blood volume. This constitutes the platelet-rich concentrate. Concentration of major platelet growth factors i.e., PDGF-AA, PDGF-AB, PDGF-BB and TGF-β1 in PRC was determined using ELISA (USCN, Schilde, Belgium) according to the manufacturer’s instructions to verify that the medium qualified as PRC. In our preparation, the concentration of PDGF-AA, PDGF-AB, PDGF-BB and TGF-β1 was found to be 50.32 ng/mL, 9.91 ng/mL, 8.32 ng/mL and 15.17 ng/mL respectively. These results were previously reported elsewhere (Shani et al., 2014).

In vitro cell viability assay

MSC viability assay was performed to verify that PRC alone could expand and sustain cell viability. Human MSCs were seeded in 24-well culture plates at a density of 1.5 × 10³ cells/well. After 48 hours of culture in growth medium, the cells were subjected to serum reduction of 1% FBS to arrest cell cycle progression for 24 hours. The cells were then either supplemented with normal growth medium containing L-DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamax-1 (all from Invitrogen-Gibco), which served as the control, or, the FBS was replaced with PRC. Our previous study indicated that 15% PRC resulted in optimal cell viability (Shani et al., 2014), and hence, the serum free medium was supplemented with 15% PRC in the experimental wells. Media was changed every 8 days and fresh PRC was also supplemented every 8 days. Cell viability was observed at 0, 8, 16 and 24 days using alamarBlue^® assay (Life Technologies, CA, USA) according to the manufacturer’s protocol. All experiments were performed in triplicate and repeated three times.

In vitro cell differentiation

Approximately 800 cells/cm² were cultured in 24-well plates and the media were either supplemented with 10% FBS (negative control) or osteogenic medium (StemPro^®, Invitrogen) (positive control) or 15% PRC. The commercially available osteogenic medium is serum free but contains dexamethasone, ascorbic acid and growth factors known to induce osteogenic differentiation. In this study, 10% FBS was used as the negative control as the supplements in FBS aid only in maintaining the cell viability and proliferation, but they do not significantly induce cell differentiation.

Quantitative real-time PCR

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to compare the relative expression of osteogenic genes. After 0, 8, 16 and 24 days, cells were detached using TypLE™(Invitrogen) express and cell pellets were lysed with buffer RLT (Qiagen, CA, USA). Total RNA was then isolated from the cells using RNeasy Mini Kit (Qiagen) in accordance with the manufacturer’s recommendations, and quantified with a nanophotometer (Implen GmbH, Germany). One µg of RNA was used to generate cDNA using QuantiTect Reverse Transcription kit (Qiagen) in accordance with the manufacturer’s instructions. Real-time PCR analysis (CFX96 Real-time system, Bio-Rad) was performed to assess the mRNA levels using QuantiTect SYBR^® Green PCR Kits (Qiagen). Each Q-PCR was performed four times for PCR yield validation. Data were analyzed by the 2^−ΔΔCt method, with normalization by the Ct of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results were expressed as relative fold changes to the gene expression levels of hMSC cultured in FBS medium.

The primers used for Q-PCR are summarized in Table 1.

Immunocytochemistry

Immunocytochemistry for osteogenic markers Runx2, osteocalcin and osteopontin were performed at days 8, 16 and 24. Briefly, MSCs were fixed in 4% paraformaldehyde for 15 min, and was then blocked for 30 min using hydrogen peroxidase (H₂O₂) to prevent endogenous activity. Cells were then incubated in goat serum working solution for 15 min to block non-specific binding followed by the primary antibody (mouse anti-human monoclonal antibody, 1:100 dilution, Abcam, Cambridge, UK) at room temperature for 30 min. After washing with PBS, cells were incubated with goat anti-mouse secondary antibody tagged with Alexa fluoro (Abcam) (1:200 dilution) for 30 min. Cells were then washed with PBS and the nucleus was counterstained with Hoechst stain (NucBlue^® Live ReadyProbes^® Reagent; Life technologies) and incubated for 15 min. After washing with PBS, it was examined under the fluorescent microscope (Nikon Eclipse TE2000-S; Nikon, Tokyo, Japan).

ALP and osteocalcin assay

For the biochemical assays, the cells were lysed with RIPA buffer. The samples were then centrifuged at 10,000× g for 5 min, and the clear supernatants were used (Gao et al., 2014). Cellular alkaline phosphates enzyme (ALP) activity, an early marker of osteogenic differentiation, was assessed at days 8, 16 and 24. ALP activity was determined colorimetrically by incubating the cell lysates with the substrate pNPP (p-nitrophenyl phosphate) (BioVision, CA, USA) in a 96-well plate at 37 °C for 60 min. ALP assay involves the dephosphorylation of pNPP by ALP to a yellow coloured pNP (p-nitrophenol), the absorbance of which was measured at 405 nm. The enzyme activity was normalized with total DNA content and expressed as nmol/ng.

Quantification of osteocalcin in cell lysates was performed at days 8, 16 and 24 and in accordance with the manufacturer’s instructions using the Gla-type Osteocalcin EIA kit (TAKARA, Shiga, Japan). The absorbance was measured at 450 nm in duplicates. The amount of osteocalcin was determined by interpolating the values from a standard curve of known concentrations. The enzyme activity was normalized with total DNA content and expressed as ng/ng. Total DNA was assessed in digested samples using a Quant-iT PicoGreen dsDNA kit according to manufacturer’s instructions (Molecular Probes, Invitrogen).

Alizarin red staining and quantification

A quantitative Alizarin Red S method was used at 8, 16 and 24 days to detect calcium compounds deposited in the extracellular matrix (ECM) as a result of bone mineralization. The cells were later fixed in 4% paraformaldehyde for 20 min. The fixed cultures were stained with 2% Alizarin Red S (Sigma-Aldrich), pH 4.2 for 10 min. Stained monolayers were visualized under the microscope (Nikon Eclipse). Quantification of the stain was performed with cetylpyridinium chloride (CPC) (Sigma-Aldrich). Briefly, 10% (w/v) cetylpyridinium chloride was prepared in PBS (pH 7.0). Stained monolayers were incubated with 1 mL of CPC with shaking for 15 min. 200 µL aliquots of the extracted dye was then transferred to 96 well plates and absorbance was measured at 570 nm (Frazier et al., 2013; Lee et al., 2014a).

Statistical analysis

Values are expressed as mean ± standard deviation. The differences between groups were analyzed using a non-parametric test (Kruskal–Wallis). If values were significant, Mann Whitney U tests were performed to determine the level of significance between the groups. Differences were considered to be significant at p ≤ 0.05. Data were analyzed using SPSS software version 17.0 (IBM Corp., Armonk, NY, USA).

Platelet yield and its effect on proliferation of hMSC

Our PRC preparation yielded approximately four fold higher platelet concentration (1157 ± 92.37 × 10³ platelets/µL) than that in the whole blood (263.71 ± 22.83 × 10³ platelets/µL) (p = 0.021). Our results also showed that PRC supplemented in serum free medium significantly increased the cell number by day 8 and maintained the cell viability for the remaining period of study, as shown in Fig. 1.

Gene expression of osteogenic markers in hMSC cultured in PRC-supplemented media

The temporal pattern of expression of osteogenic gene markers in the PRC group was similar to that in the osteogenic medium throughout the duration of culture; however, the expression of certain osteogenic genes were significantly higher in the PRC group compared to the osteogenic medium at specific time points (Fig. 2). At the early time point (day 8), PRC upregulated the expression of transcription factor Runx2 by 3-fold and collagen type I (Col 1) by 2-fold compared to the osteogenic medium. The expression of ALP was 1-fold higher in the PRC group compared to the osteogenic medium (p < 0.05) at day 16. By day 24, expression of bone morphogenetic protein 2 (BMP2), one of the key factors in osteogenesis, increased by approximately 6-fold in the PRC group compared to the osteogenic medium (p < 0.05). The expression of another ECM marker, osteopontin, produced by the osteoblasts was also significantly higher by 5-fold at day 16 and 4-fold at day 24 in the PRC group compared to the osteogenic medium. On the other hand, throughout the duration of cell culture, the expression of osteonectin, a late osteogenic marker, seemed to be persistently upregulated in the OM compared to the PRC supplement (p < 0.05).

Immunocytochemical staining of osteogenic markers

Immunocytochemical analysis showed that in comparison with cells cultured in the osteogenic medium, cells treated with PRC had stronger staining for Runx2 (Fig. 3A) and comparable staining intensity for both Osteocalcin (Fig. 3B) and Osteopontin (Fig. 3C). Staining for all three markers were absent in cells cultured in the FBS-containing medium.

ALP and osteocalcin assays

There was a significant increase in ALP activity of cells in both the PRC and osteogenic medium (p < 0.05) compared to the cells in the FBS medium (Fig. 4A). The level appeared to be slightly higher in the PRC group compared to that in the osteogenic medium at day 8 and 16 (p < 0.05). There was a gradual increment of osteocalcin protein expression throughout the duration of cell culture. Similar to the ALP activity, osteocalcin level was also significantly higher in cells cultured in PRC and osteogenic medium compared to the control group (p < 0.05) (Fig. 4B). However, cells in the PRC group had a higher level of osteocalcin compared to the osteogenic medium (p < 0.05).

Alizarin red staining and quantification

The mineralized large nodules formed by cells cultured in PRC were intensely stained with Alizarin red S stain, indicating a higher degree of mineralization compared to the control group (Fig. 5A). Throughout the course of the study, there was a gradual increase in the absorbance of the Alizarin Red S stain (quantified by the extraction of the calcified mineral from the stained monolayer) (Fig. 5B), which is indicative of bone mineralization in the differentiated cells. PRC group had significantly higher bone mineralization compared to the control group. Its level was comparable to that of the osteogenic medium, indicating that PRC was equally effective in inducing mineralization as the osteogenic medium.