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Open Access Research article

The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells

John Antoniou12, Hong Tian Wang2, Abdulrahman M Alaseem12, Lisbet Haglund3, Peter J Roughley4 and Fackson Mwale12*

Author Affiliations

1 Division of Orthopaedic Surgery, McGill University, 1650 Cedar Avenue, Montreal, QC, H3G 1A4, Canada

2 Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, 3755 Chemin de la Cote Ste-Catherine, Montreal, QC, H3T 1E2, Canada

3 Orthopaedic Research Laboratory, Royal Victoria Hospital, McGill University, 687 Pine Avenue West Montreal, QC, H3A 1A1, Canada

4 Genetics Unit, Shriners Hospitals for Children, 1529 Cedar Avenue, Montreal, QC, H3G 1A6, Canada

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Arthritis Research & Therapy 2012, 14:R267  doi:10.1186/ar4113


The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/14/6/R267


Received:28 June 2012
Revisions received:16 November 2012
Accepted:5 December 2012
Published:10 December 2012

© 2012 Antoniou et al.; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

We previously showed that Link N can stimulate extracellular matrix biosynthesis by intervertebral disc (IVD) cells, both in vitro and in vivo, and is therefore a potential stimulator of IVD repair. The purpose of the present study was to determine how Link N may influence human mesenchymal stem cell (MSC) differentiation, as a prelude to using Link N and MSC supplementation in unison for optimal repair of the degenerated disc.

Methods

MSCs isolated from the bone marrow of three osteoarthritis patients were cultured in chondrogenic or osteogenic differentiation medium without or with Link N for 21 days. Chondrogenic differentiation was monitored by proteoglycan staining and quantitation by using Alcian blue, and osteogenic differentiation was monitored by mineral staining and quantitation by using Alzarin red S. In addition, proteoglycan secretion was monitored with the sulfated glycosaminoglycan (GAG) content of the culture medium, and changes in gene expression were analyzed with real-time reverse transcription (RT) PCR.

Results

Link N alone did not promote MSC chondrogenesis. However, after MSCs were supplemented with Link N in chondrogenic differentiation medium, the quantity of GAG secreted into the culture medium, as well as aggrecan, COL2A1, and SOX9 gene expression, increased significantly. The gene expression of COL10A1 and osteocalcin (OC) were downregulated significantly. When MSCs were cultured in osteogenic differentiation medium, Link N supplementation led to a significant decrease in mineral deposition, and alkaline phosphatase (ALP), OC, and RUNX2 gene expression.

Conclusions

Link N can enhance chondrogenic differentiation and downregulate hypertrophic and osteogenic differentiation of human MSCs. Therefore, in principle, Link N could be used to optimize MSC-mediated repair of the degenerated disc.

Introduction

Intervertebral disc (IVD) degeneration plays a major role in the etiology of low-back pain (LBP), which can significantly affect more than half of the population at some point during their lives [1-3]. Disc degeneration usually is associated with increased breakdown of matrix, decreased synthesis of aggrecan and collagen, cell loss through apoptosis, and cluster formation by surviving cells [4-7]. It is evident that vascular ingrowth can occur into the degenerated IVD, and that in painful degenerated IVDs, the vessels are accompanied by nociceptive nerves [1]. Thus, reversing the degeneration process and repairing the degenerated IVDs is important for LBP therapy.

Disc repair may be enhanced by cell supplementation to maximize extracellular matrix production. The source of cells for disc repair is not immediately apparent, as no benign site is known for harvesting authentic autologous disc cells, and it is preferable to avoid the use of allogeneic donor disc cells. Although the avascular nature of the disc may make the nucleus pulposus (NP) an immunologically privileged site, and therefore make the use of allogeneic cells a tempting proposition, the risk of transferring infectious agents remains real. Thus, we must generate disc cells from another autologous source.

One possibility is to use mesenchymal stem cells (MSCs). MSCs are multipotent stem cells that can be isolated, expanded, and stimulated to differentiate into a variety of cells, including osteoblasts, chondrocytes, myocytes, adipocytes, and beta pancreatic islets cells [8-11]. Recent studies have shown that MSCs can be used in biologic repair of cartilage or IVD lesions, because of the bioactive factors secreted by MSCs and the proliferation of chondrocytes differentiated from MSCs [12-18]. MSCs can be injected directly, or together with a scaffold, into the degenerated disc, where they can differentiate into disc cells, produce extracellular matrix (ECM), and reestablish healthy disc function [19-22]. It is well known that growth factors, such as bone morphogenetic proteins (BMPs) and transforming growth factor-β (TGF-β) can be applied directly for tissue regeneration. Injecting MSCs together with growth factors into IVDs can bring added benefits to the repair process, considering that growth factors and MSCs can improve tissue repair individually, and growth factors can stimulate and accelerate the differentiation of MSCs to chondrocytes.

Hyaline cartilage and healthy NP possess similar macromolecules in their extracellular matrix [23-25], although some differences in the structure of proteoglycans in cartilage and the NP have been observed [26]. The production of an extracellular matrix with a high proteoglycan-to-collagen ratio can distinguish NP cells from chondrocytes and could help in identifying an NP-like phenotype in vivo, as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc [27].

As an economical alternative to growth factors, it may be possible to use Link N together with MSCs for tissue regeneration. Link N (DHLSDNYTLDHDRAIH) is the N-terminal peptide of link protein, a glycoprotein that stabilizes the non-covalent interaction between an aggrecan G1 domain and hyaluronate. Link N can stimulate the synthesis of collagen in human articular cartilage and bovine IVD cells in vitro [28-30], as well as in the IVDs of rabbits in vivo [31]. Our previous results showed that Link N can also decrease the expression of type X collagen, a marker of chondrocyte hypertrophy [32], and stimulate the expression of type II collagen, a marker of cartilage and disc ECM [33], in human mesenchymal stem cells [34]. Thus, Link N has the potential to be used together with MSCs in promoting the formation of the type of ECM necessary for IVD repair. However, to be useful for this purpose, it is essential that Link N does not interfere with MSC chondrogenesis.

The purpose of this research was to determine the effects of Link N on human MSCs cultured in chondrogenic and osteogenic differentiation media to determine how Link N affects these processes.

Materials and methods

Source and isolation of stem cells

MSCs were obtained from aspirates of the intramedullary canal of three osteoarthritis patients (aged 40 to 60 years) undergoing total hip replacement, with a protocol approved by the Research Ethics Committee of the Jewish General Hospital (Montreal, QC, Canada). We had all necessary consent from any patients involved in the study, including consent to participate in the study and consent to publish. Bone marrow aspirates were processed as previously described [34]. In brief, each aspirate was diluted 1:1 (vol/vol) with Dulbecco Modified Eagle Medium (DMEM) containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Wisent Inc., St-Bruno, QC, Canada) and then layered over Ficoll (Ficoll-Paque Plus; GE Healthcare Bio-Sciences, Baie-d'Urfé, QC, Canada). After centrifugation at 900 g for 30 minutes, the mononuclear cell layer was removed from the interface, washed with DMEM, and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were plated in culture dishes and incubated at 37°C in a humidified atmosphere with 5% CO2. After 48 hours, nonadherent cells were washed off, and the adherent cells were thoroughly washed twice with DMEM. All cells were expanded in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and were used within four passages.

Cell culture

In every well of a 24-well plate, about 4,000 MSCs were plated and cultured in expansion medium, high glucose DMEM with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Floating cells were removed after allowing MSCs to attach overnight, and then the attached MSCs were cultured in 1 ml of the same medium for 3 days. The cells were washed with phosphate-buffered saline (PBS) 3 times, and then were cultured in chondrogenic or osteogenic differentiation medium. Link N was dissolved in the differentiation media at a final concentration of 0.1 μg/ml or 1.0 μg/ml. Differentiation medium without Link N was used as a control. The medium was changed every 3 days. The used chondrogenic differentiation media were stored at -20°C for GAG analysis.

For gene expression analysis, 5 × 105 MSCs were plated on 35 × 10-mm dishes (Sarstedt, Quebec, QC, Canada). After the cells were cultured in expansion medium (see earlier) for 3 days, they were cultured in chondrogenic or osteogenic differentiation medium with the same conditions as earlier.

To compare the direct effects of Link N and TGF-β3 on MSC chondrogenesis, MSCs were cultured in serum-free medium, serum-free medium with 1 μg/ml Link N, or serum-free medium with 10 ng/ml TGF-β3 for 21 days. Serum-free medium was prepared with high-glucose DMEM with 1 × ITS+1 premix (Sigma-Aldrich, Ontario, QC, Canada), 100 nM dexamethasone, 50 μg/ml ascorbic acid-2-phosphate, 40 μg/ml proline, 100 units/ml penicillin, and 100 μg/ml streptomycin.

Chondrogenic differentiation and proteoglycan analysis

Chondrogenic differentiation medium containing TGF-β was from Invitrogen (Burlington, ON, Canada). Chondrogenic differentiation was monitored by proteoglycan accumulation by using Alcian blue staining [35]. In brief, after the cells were cultured for 21 days, the culture medium was removed, the cells were rinsed gently with PBS twice, and then fixed with -20°C methanol for 30 minutes. After rinsing with PBS twice, the wells were stained with Alcian blue in 0.1N HCl for 30 minutes. The wells were then rinsed with distilled water 3 times; and images of stained wells were captured. To quantify proteoglycan, the matrix-associated dye was extracted with 6 M guanidine-HCl (Sigma-Aldrich, Oakville, ON, Canada; 200 μl/well) and measured at 620 nm.

Osteogenic differentiation and mineralization analysis

The osteogenic differentiation medium was prepared with high-glucose DMEM containing 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, 50 μM L-ascorbic acid, 100 units/ml penicillin, and 100 μg/ml streptomycin. Osteogenic differentiation was monitored by mineral deposition by using alizarin red S staining [36]. In brief, after the cells were cultured for 21 days, the medium was removed, and the cells were rinsed once with distilled water. The cells were then fixed with 70% ethanol (stored at -20°C) for 30 minutes. After fixation, the wells were rinsed twice with distilled water, and the cells were stained with 2% alizarin red S (pH 4.2) for 30 minutes. Finally, the wells were rinsed 3 times with distilled water, and images of stained wells were captured. To quantify the matrix mineralization, alizarin red S was extracted with 100 mM cetylpyridinium chloride (Sigma-Aldrich; 1 ml/well) and measured at 570 nm.

GAG and DNA analysis

The sulfated glycosaminoglycan (GAG) content of the culture medium was detected by using a 96-well round-bottom plate [37]. To 20 μl culture medium in every well, 180 μl of 16 mg/L 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich) solution was added. The absorbance of the solution was monitored immediately at 535 nm. Chondroitin 6-sulfate from shark cartilage was used as a standard. DNA was measured on days 3 and 21 by using a Quant-iT dsDNA High-Sensitivity Assay Kit (Invitrogen, Burlington, ON, Canada), following the manufacturer's instructions. In brief, 20 μl of DNA solution was mixed with 200 μl of working solution in each well of a 96-well plate. Fluorescence measurements were taken with an excitation wavelength of 480 nm and emission wavelength of 530 nm. A standard curve was obtained from λ DNA.

Total RNA isolation

Cells were harvested at days 7, 14, and 21 for gene-expression studies. Total RNA was extracted from MSCs by using Trizol (Invitrogen), following the manufacturer's instructions. After centrifugation for 15 minutes at 12,000 g at 4°C, the aqueous phase was precipitated with 1 volume of isopropanol, incubated for 45 minutes at -20°C, and centrifuged again for 15 minutes at 12,000 g at 4°C. The resulting RNA pellet was washed with 75% ethanol, centrifuged, and air-dried. The pellets were dissolved in 50 μl diethylpyrocarbonate (DEPC)-treated distilled water and assayed for RNA concentration, by measuring A260, and purity, by calculating the A260/A280 ratio.

Reverse transcription

One microgram total RNA isolated from the cells was digested with DNase I (Invitrogen). Then, 1 μg RNA was mixed with random primers (final concentration, 0.15 μg/μl), dNTP mixture (final concentration 0.5 mM), and DEPC-treated distilled water to a total volume of 12 μl. Following the instructions of the reagent supplier (Invitrogen), the solution was incubated at 65°C for 5 minutes, and then it was mixed with reaction buffer, DTT, RNaseOUT, and Superscript II reverse transcriptase with a final volume of 20 μl. The solution was incubated at 45°C for 50 minutes and then at 70°C for 15 minutes to inactivate the reverse transcriptase.

Real-time PCR

For LightCycler real-time PCR, a master mix of the following reaction components was prepared: 10 μl SYBER PCR master mix (×1) (Qiagen, Mississauga, ON, Canada), 8 μl distilled water, 0.5 μl forward primer (0.25 μM), and 0.5 μl reverse primer (0.25 μM). The nucleotide sequences of the primers are listed in Table 1. To each 19 μl master mix, 1 μl of cDNA was added as the PCR template. The PCR conditions included one cycle of initial activation (95°C for 15 minutes, 20°C/s ramp rate), 45 cycles of amplification and quantification (94°C for 15 seconds, 57°C for 30 seconds, 72°C for 30 seconds), one cycle of melting-curve determination (65°C to 95°C with heating rate of 0.1°C per second, with a continuous fluorescence measurement), and final cooling to 4°C.

Table 1. Primer sequences

The crossing points (CPs) were determined by the LightCycler software 3.3 (Roche Diagnostics, Indianapolis, IN, USA) and were measured at constant fluorescence level. The ratio of gene expression relative to GAPDH as the reference gene was determined by the following equation [38]:

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Statistical analysis

Statistical analysis was performed by using analysis of variance followed by Fisher protected least significant difference post hoc test by using Statview (SAS Institute Inc., Cary, NC, USA). Results are presented as the mean ± standard deviation (SD) of three independent experiments with cells from three different donors. Differences were considered statistically significant with P < 0.05.

Results

Analysis of MSCs cultured in chondrogenic differentiation medium with or without Link N demonstrated differentiation into a chondrogenic lineage by Alcian blue staining (Figure 1A). When Alcian blue was extracted from the stained cultures (Figure 1B), no significant differences in proteoglycan deposition were found between control cells and cells cultured with Link N.

thumbnailFigure 1. Effect of Link N on proteoglycan deposition by mesenchymal stem cells (MSCs) in chondrogenic differentiation medium. (A) Alcian blue staining of proteoglycan in extracellular matrix after MSCs were cultured in chondrogenic differentiation medium without (control) or with Link N for 21 days. (B) The absorbance value of the solubilized Alcian blue at 620 nm. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors.

Proteoglycan production during chondrogenic differentiation can also be monitored with GAG analysis, and this was used to assess the effect of Link N on MSC differentiation. After MSCs were cultured in chondrogenic differentiation medium with Link N for 9, 12, and 15 days, the quantity of GAG secreted into the culture medium was significantly higher than that in control medium (Figure 2). This difference was not observed at days 3, 6, 18, and 21, although a tendency to increased GAG synthesis with Link N supplementation was observed on days 6, 18, and 21. Because the DNA content of the cultures did not change significantly between days 3 and 21 of culture, the enhanced GAG synthesis is likely the result of increased production by each cell rather than a consequence of having more cells because of cell proliferation.

thumbnailFigure 2. Glycosaminoglycan (GAG) secretion into 1-ml culture medium over the 3-day period preceding medium collection when mesenchymal stem cells (MSCs) were cultured in chondrogenic differentiation medium in the absence (control) or presence of Link N. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors. *P < 0.05 versus control.

Aggrecan (ACAN) and COL2A1 are two important genes that define the chondrocyte phenotype [4,5], and SOX9 is an important transcription regulator of chondrogenesis [39,40]. The effect of Link N on the expression of these genes was therefore assessed. After the cells were cultured for 7 days in chondrogenic differentiation medium, ACAN expression with 0.1 μg/ml or 1.0 μg/ml Link N increased significantly compared with controls (P = 0.001 and 0.002, respectively) (Figure 3A). The increase was also observed after 14 days (P = 0.003 for 0.1 μg/ml Link N; P = 0.004 for 1.0 μg/ml Link N). Similarly, COL2A1 expression was also increased compared with control cells at both day 7 (P = 0.003 for 0.1 μg/ml Link N; P = 0.005 for 1.0 μg/ml Link N) and day 14 (P = 0.003 for 0.1 μg/ml Link N, P = 0.002 for 1.0 μg/ml Link N) (Figure 3B). Compared with control cells, the expression of SOX9 was increased at day 7 (P = 0.017 for 0.1 μg/ml Link N; P = 0.027 for 1.0 μg/ml Link N) and day 14 (P = 0.024 for 0.1 μg/ml Link N; P = 0.011 for 1.0 μg/ml Link N) (Figure 3C). For the expression of all three genes, no significant difference was observed between the two concentrations of Link N. Thus Link N appeared to promote the chondrogenic differentiation of MSCs.

thumbnailFigure 3. Relative ratio of ACAN (A), COL2A1 (B), and SOX9 (C) gene expression in mesenchymal stem cells (MSCs) cultured in chondrogenic differentiation medium without (control) or with 0.1 or 1.0 μg/ml Link N for 7, 14, and 21 days. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors. *P < 0.05 versus control.

COL10A1 is a marker gene for hypertrophic chondrocyte differentiation, a process that is undesirable for effective disc or cartilage repair [41,42]. When MSCs were cultured in chondrogenic differentiation medium with 1.0 μg/ml Link N for 7 (P = 0.002), 14 (P = 0.001) and 21 days (P = 0.007), gene expression was downregulated significantly (Figure 4A). When MSCs were cultured in chondrogenic differentiation medium with 0.1 μg/ml Link N for 7 and 14 days, no significant difference of COL10A1 expression was observed compared with the control cells. However, a significant difference was observed at day 21 (P = 0.015). The difference between cells cultured in different concentrations of Link N was significant at day 7 (P = 0.006). Thus, Link N appears to reduce hypertrophic differentiation by the MSCs.

thumbnailFigure 4. Relative ratio of COL10A1 (A) and OC (B) gene expression in mesenchymal stem cells (MSCs) cultured in chondrogenic differentiation medium without (control) or with 0.1 or 1.0 μg/ml Link N. The expression of COL10A1 was detected after the cells were cultured for 7, 14, or 21 days. The expression of OC was detected after the cells were cultured for 21 days. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors. *P < 0.05 versus control.

Because another major problem with MSC-based chondrogenesis is their osteogenic differentiation, we next explored the effect of Link N on osteocalcin (OC), an important osteogenic marker [43,44]. When MSCs were cultured in chondrogenic differentiation medium with 0.1 μg/ml and 1.0 μg/ml Link N for 21 days, the expression of OC was significantly downregulated (P < 0.001) (Figure 4B).

To determine whether Link N can directly stimulate chondrogenesis, MSCs were cultured in serum-free medium without (control) or with either Link N or TGF-β3. Proteoglycan deposition in the accumulated ECM was visualized by staining with Alcian blue (Figure 5A). When Alcian blue was extracted from the stained cultures, only TGF-β3 supplementation led to increased proteoglycan deposition when compared with the control cells and those cultured with Link N (Figure 5B). Thus, Link N alone does not directly stimulate chondrogenesis in a manner analogous to TGF-β, but rather enhances ongoing chondrogenesis.

thumbnailFigure 5. Effect of Link N on proteoglycan deposition by mesenchymal stem cells (MSCs) in serum-free medium. (A) Alcian blue staining of proteoglycan in extracellular matrix after MSCs were cultured in serum-free medium devoid of TGF-β (control), serum-free medium with 1 μg/ml Link N, and serum-free medium with 10 ng/ml TGF-β3 for 21 days. (B) The absorbance value of the solubilized Alcian blue at 620 nm. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors.

After MSCs were cultured in osteogenic differentiation medium without or with 0.1 or 1.0 μg/ml Link N for 21 days, mineral deposition in the accumulated ECM was visualized by staining with alizarin red S (Figure 6A). With the higher concentration of Link N, a decrease appeared to exist in the extent of matrix mineralization. When alizarin red S was extracted from the matrix, mineral deposition in the presence of 1.0 μg/ml Link N was shown to be decreased significantly compared with control cells (Figure 6B).

thumbnailFigure 6. Effect of Link N on mineral deposition by mesenchymal stem cells (MSCs) in osteogenic differentiation medium. (A) Alizarin red staining of mineral deposition in the extracellular matrix after MSCs were cultured in osteogenic differentiation medium without (control) or with Link N for 21 days. (B) The absorbance value of solubilized alizarin red measured at 620 nm. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors. *P < 0.05 versus control.

Alkaline phosphatase (ALP) and osteocalcin (OC) are important genes to define the osteogenic phenotype [43-45], and RUNX2 is an important transcription regulator of osteogenesis [45,46]. The effect of Link N on the expression of these genes was therefore assessed. After the cells were cultured in osteogenic differentiation medium with 1.0 μg/ml Link N for 7 days, ALP (P = 0.017), OC (P = 0.016), and RUNX2 (P = 0.019) expression decreased significantly compared with that in control cells (Figure 7A through C). No obvious effect was detected with 0.1 μg/ml Link N. At day 21, no significant difference of ALP, OC, and RUNX2 expression was observed compared with control cells for either 0.1 or 1.0 μg/ml Link N. Thus, at the higher concentration, Link N appeared to downregulate the osteogenic differentiation potential of the MSCs.

thumbnailFigure 7. The relative ratio of ALP (A), OC (B), and RUNX2 (C) gene expression in mesenchymal stem cells (MSCs) cultured in osteogenic differentiation medium without (control) or with 0.1 or 1.0 μg/ml Link N for 7 and 21 days. The results are shown as mean ± SD of three independent experiments with MSCs from three different donors. *P < 0.05 versus control.

Discussion

If Link N is to be used to stimulate disc repair in the presence of MSCs, it is essential that it does not interfere with chondrogenic differentiation and, ideally, promotes it. The results of the present work indicate that Link N can induce the expression of aggrecan and type II collagen, possibly through activation of SOX9 expression, and that the upregulation of aggrecan gene expression can increase proteoglycan production. Aggrecan and type II collagen are two important components of the extracellular matrix in IVD, and their degradation is associated with IVD degeneration [4,5,47]. Restoration of aggrecan and type II collagen levels within the disc ECM is therefore an essential requisite for disc repair. Thus Link N has the potential to be used together with MSCs in repairing degenerated IVDs.

It should be noted that although in vitro chondrogenesis is performed mostly in either pellet cultures or in a 3D environment, such as a hydrogel, the experiments presented here were not performed under these conditions. Monolayer culture was preferred, as it better mimics how stem cells will deposit on the disc ECM if they are to be injected in vivo for biologic repair. Future studies will be conducted to determine whether injection of Link N together with MSCs in an animal model of disc degeneration or into degenerated human discs in organ culture can promote ECM production.

A key consideration in using MSCs for repairing degenerated IVDs is not only the promotion of chondrogenesis, but also the prevention of hypertrophy and osteogenesis [48,49]. In the present work, such a process is indicated by decreased gene expression of type X collagen, a marker for hypertrophy, as well as ALP and OC, two important bone-matrix proteins synthesized by osteoblasts, and by decreased matrix mineralization through calcium deposition [50]. The ability of Link N to decrease the expression of ALP and OC may be through downregulating the transcription regulator RUNX2. Thus Link N can both favor chondrogenic differentiation of MSCs and retard their hypertrophic and osteogenic differentiation.

In the present study, it was not our intention to address directly the ability of Link N to promote chondrogenic, osteogenic, or disc-like differentiation. The goal of the work was to check that Link N does not negatively interfere with chondrogenesis, a differentiation pathway common to disc cells, which make matrix molecules common to all cartilages. Stem cell conversion to an NP-like phenotype rather than a hyaline cartilage is distinguished by the production of a gelatinous matrix with a proteoglycan-to-collagen ratio of approximately 28:1 [27]. When Link N is used in vivo, it is expected that growth factors such as TGF-β will always be present, and that these, coupled with the unique extracellular milieu, will guide the formation of either a disc-like or a cartilage-like matrix.

The influences of Link N on stimulating chondrogenesis and decreasing osteogenesis are similar to those reported with BMP and TGF-β [51,52]. However, one major advantage of Link N over a growth factor such as TGF-β for therapeutic use is the large saving in cost. For example, Link N costs $750 for 50 mg of the synthetic peptide, which is about 2 cents per microgram for supplementation. In contrast, TGF-β3 costs $30 per microgram. Link N therefore represents an economic therapeutic agent with potentially beneficial effects on disc repair when used either alone or in the presence of MSCs.

Previous studies have shown that Link N can stimulate the synthesis of proteoglycans and collagens in both articular cartilage and IVD [28-30]. The present data indicate that Link N and MSCs can synergize to stimulate chondrogenesis while suppressing osteogenesis. These are features needed not only for any agent designed to stimulate disc repair but also for any agent designed to stimulate articular cartilage repair. Therefore, in principle, disc and cartilage repair may be enhanced by stem cell supplementation in the presence of Link N to maximize extracellular matrix production. Such a beneficial effect does, however, assume that there is no nutrient deprivation that could impair cell metabolism. Although this could be a potential impediment to disc repair because of its avascular nature and the lack of a bathing nutrient fluid, it is less likely to be a detriment to articular cartilage repair where the tissue is bathed in synovial fluid and nutrient-diffusion distances are relatively short. Only future in vivo studies will be able to determine whether biologic repair of the degenerate disc by Link N and MSC supplementation could be a reality.

The present data indicate that Link N alone cannot directly promote MSC chondrogenesis. Thus, together with the other data, it appears that Link N enhances ongoing chondrogenesis rather than initiates it. Furthermore, Link N appears to be capable of reducing hypertrophic differentiation of chondrocytes. These results have implications in relation to regenerating a functional nucleus pulposus in the degenerated IVD or in repairing cartilage.

Conclusions

In addition to promoting chondrogenesis and stimulating the gene expression of aggrecan and type II collagen, Link N also is able to downregulate osteogenesis and the gene expression oftype X collagen, alkaline phosphatase, and osteocalcin. Interestingly, these properties of Link N are similar to those reported previously for several growth factors and are features needed for any biologic agent designed to stimulate disc or cartilage repair. Therefore, in principle, Link N and MSC supplementation could have therapeutic value for the treatment of degenerative lesions in both disc and articular cartilage.

Abbreviations

ACAN: aggrecan; ALP: alkaline phosphatase; COL2A1: collagen, type 2, alpha 1; DMMB: 1,9-dimethylmethylene blue; ECM: extracellular matrix; GAG: sulfated glycosaminoglycan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IVD: intervertebral disc; MSC: mesenchymal stem cell; NP: nucleus pulposus; OC: osteocalcin; PCR: polymerase chain reaction; RUNX2: Runt-related transcription factor 2; SOX9: SRY (sex-determining region Y)-box 9.

Competing interests

All the authors declare that they have no competing interests.

Authors' contributions

JA supervised the research. HTW performed the experiments, data acquisition, and statistical analysis, as well as manuscript writing. AMA contributed to the analysis of osteogenesis by real-time RT-PCR. LH and PJR made substantial contributions to the study design and revised the manuscript. FM conceived and supervised the whole study and finished writing the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The Canadian Institutes of Health Research (CIHR), the AO Foundation, and the North American Spine Society (NASS) supported the study.

References

  1. Freemont AJ: The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain.

    Rheumatology 2009, 48:5-10. PubMed Abstract | Publisher Full Text OpenURL

  2. Smith LJ, Nerurkar NL, Choi KS, Harfe BD, Elliott DM: Degeneration and regeneration of the intervertebral disc: lessons from development.

    Dis Model Mech 2011, 4:31-41. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  3. Miller JA, Schmatz C, Schultz AB: Lumbar disc degeneration: correlation with age, sex and spine level in 600 autopsy specimens.

    Spine 1988, 13:173-178. PubMed Abstract | Publisher Full Text OpenURL

  4. Adams MA, Roughley PJ: What is intervertebral disc degeneration, and what causes it?

    Spine 2006, 31:2151-2161. PubMed Abstract | Publisher Full Text OpenURL

  5. Roughley PJ: Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix.

    Spine 2004, 29:2691-2699. PubMed Abstract | Publisher Full Text OpenURL

  6. Roughley PJ, Melching LI, Heathfield TF, Pearce RH, Mort JS: The structure and degradation of aggrecan in human intervertebral disc.

    Eur Spine J 2006, 15(suppl 3):326-332. OpenURL

  7. Le Maitre CL, Pockert A, Buttle DJ, Freemont AJ, Hoyland JA: Matrix synthesis and degradation in human intervertebral disc degeneration.

    Biochem Soc Trans 2007, 35:652-655. PubMed Abstract | Publisher Full Text OpenURL

  8. Caplan AI, Bruder SP: Mesenchymal stem cells: building blocks for molecular medicine in the 21st century.

    Trends Mol Med 2001, 7:259-264. PubMed Abstract | Publisher Full Text OpenURL

  9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells.

    Science 1999, 284:143-147. PubMed Abstract | Publisher Full Text OpenURL

  10. Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues.

    Science 1997, 276:71-74. PubMed Abstract | Publisher Full Text OpenURL

  11. Barry F, Boynton RE, Liu B, Murphy JM: Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components.

    Exp Cell Res 2001, 268:189-210. PubMed Abstract | Publisher Full Text OpenURL

  12. Baksh D, Song L, Tuan RS: Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.

    J Cell Mol Med 2004, 8:301-316. PubMed Abstract | Publisher Full Text OpenURL

  13. Csaki C, Schneider PR, Shakibaei M: Mesenchymal stem cells as a potential pool for cartilage tissue engineering.

    Ann Anat 2008, 190:395-412. PubMed Abstract | Publisher Full Text OpenURL

  14. Caplan AI, Dennis JE: Mesenchymal stem cells as trophic mediators.

    J Cell Biochem 2006, 98:1076-1084. PubMed Abstract | Publisher Full Text OpenURL

  15. Liu CH, Hwang SM: Cytokine interactions in mesenchymal stem cells from cord blood.

    Cytokine 2005, 32:270-279. PubMed Abstract | Publisher Full Text OpenURL

  16. Schinkothe T, Bloch W, Schmidt A: In vitro secreting profile of human mesenchymal stem cells.

    Stem Cells Dev 2008, 17:199-206. PubMed Abstract | Publisher Full Text OpenURL

  17. Centeno CJ, Busse D, Kisiday J, Keohan C, Freeman M, Karli D: Increased knee cartilage volume in degenerative joint disease using percutaneously implanted, autologous mesenchymal stem cells.

    Pain Physician 2008, 11:343-353. PubMed Abstract | Publisher Full Text OpenURL

  18. Murphy JM, Fink DJ, Hunziker EB, Barry FP: Stem cell therapy in a caprine model of osteoarthritis.

    Arthritis Rheum 2003, 48:3464-3474. PubMed Abstract | Publisher Full Text OpenURL

  19. Crevensten G, Walsh AJ, Ananthakrishnan D, Page P, Wahba GM, Lotz JC, Berven S: Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs.

    Ann Biomed Eng 2004, 32:430-434. PubMed Abstract OpenURL

  20. Richardson SM, Hughes N, Hunt JA, Freemont AJ, Hoyland JA: Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.

    Biomaterials 2008, 29:85-93. PubMed Abstract | Publisher Full Text OpenURL

  21. Richardson SM, Hoyland JA: Stem cell regeneration of degenerated intervertebral discs: current status.

    Curr Pain Headache Rep 2008, 12:83-88. PubMed Abstract | Publisher Full Text OpenURL

  22. Leung VY, Chan D, Cheung KM: Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction.

    Eur Spine J 2006, 15(suppl 3):406-413. OpenURL

  23. Hayes AJ, Benjamin M, Ralphs JR: Extracellular matrix in development of the intervertebral disc.

    Matrix Biol 2001, 20:107-121. PubMed Abstract | Publisher Full Text OpenURL

  24. Sztrolovics R, Grover J, Cs-Szabo G, Shi SL, Zhang Y, Mort JS, Roughley PJ: The characterization of versican and its message in human articular cartilage and intervertebral disc.

    J Orthop Res 2002, 20:257-266. PubMed Abstract | Publisher Full Text OpenURL

  25. Adams P, Eyre DR, Muir H: Biochemical aspects of development and ageing of human lumbar intervertebral discs.

    Rheumatol Rehabil 1977, 16:22-29. PubMed Abstract | Publisher Full Text OpenURL

  26. Buckwalter JA, Smith KC, Kazarien LE, Rosenberg LC, Ungar R: Articular cartilage and intervertebral disc proteoglycans differ in structure: an electron microscopic study.

    J Orthop Res 1989, 7:146-151. PubMed Abstract | Publisher Full Text OpenURL

  27. Mwale F, Roughley P, Antoniou J: Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc.

    Eur Cell Mater 2004, 8:58-64. PubMed Abstract | Publisher Full Text OpenURL

  28. Liu H, McKenna LA, Dean MF: An N-terminal peptide from link protein can stimulate biosynthesis of collagen by human articular cartilage.

    Arch Biochem Biophys 2000, 378:116-122. PubMed Abstract | Publisher Full Text OpenURL

  29. Mwale F, Demers CN, Petit A, Roughley P, Poole AR, Steffen T, Aebi M, Antoniou J: A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc.

    J Cell Biochem 2003, 88:1202-1213. PubMed Abstract | Publisher Full Text OpenURL

  30. Petit A, Yao G, Rowas SA, Gawri R, Epure L, Antoniou J, Mwale F: Effect of synthetic link N peptide on the expression of type I and type II collagens in human intervertebral disc cells.

    Tissue Eng Part A 2011, 17:899-904. PubMed Abstract | Publisher Full Text OpenURL

  31. Mwale F, Masuda K, Pichika R, Epure LM, Yoshikawa T, Hemmad A, Roughley PJ, Antoniou J: The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration.

    Arthritis Res Ther 2011, 13:R120. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  32. Tchetina E, Mwale F, Poole AR: Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation.

    J Bone Miner Res 2003, 18:844-851. PubMed Abstract | Publisher Full Text OpenURL

  33. Bobick BE, Chen FH, Le AM, Tuan RS: Regulation of the chondrogenic phenotype in culture.

    Birth Defects Res C 2009, 87:351-371. Publisher Full Text OpenURL

  34. Mwale F, Demers CN, Petit A, Antoniou J: Effect of the N-terminal peptide of Link protein on human mesenchymal stem cells from osteoarthritis patients.

    J Stem Cells 2008, 3:99. Publisher Full Text OpenURL

  35. Akiyama H, Hiraki Y, Shigeno C, Kohno H, Shukunami C, Tsuboyama T, Kasai R, Suzuki F, Konishi J, Nakamura T: 1 alpha,25-Dihydroxyvitamin D3 inhibits cell growth and chondrogenesis of a clonal mouse EC cell line, ATDC5.

    J Bone Miner Res 1996, 11:22-28. PubMed Abstract | Publisher Full Text OpenURL

  36. Cash DE, Bock CB, Schughart K, Linney E, Underhill TM: Retinoic acid receptor α function in vertebrate limb skeletogenesis: a modulator of chondrogenesis.

    J Cell Biol 1997, 136:445-457. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  37. Farndale RW, Buttle DJ, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.

    Biochim Biophys Acta 1986, 883:173-177. PubMed Abstract | Publisher Full Text OpenURL

  38. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR.

    Nucleic Acids Res 2001, 29:e45. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  39. Han Y, Lefebvre V: L-Sox5 and Sox6 drive expression of the aggrecan gene in cartilage by securing binding of Sox9 to a far-upstream enhancer.

    Mol Cell Biol 2008, 28:4999-5013. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  40. Akiyama H: Control of chondrogenesis by the transcription factor Sox9.

    Mod Rheumatol 2008, 18:213-219. PubMed Abstract | Publisher Full Text OpenURL

  41. Tchetina E, Mwale F, Poole AR: Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation.

    J Bone Miner Res 2003, 18:844-851. PubMed Abstract | Publisher Full Text OpenURL

  42. Wu W, Mwale F, Tchetina E, Kojima T, Yasuda T, Poole AR: Cartilage matrix resorption in skeletogenesis.

    Novartis Found Symp 2001, 232:158-166.

    discussion 166-170

    PubMed Abstract OpenURL

  43. Shum L, Nuckolls G: The life cycle of chondrocytes in the developing skeleton.

    Arthritis Res 2002, 4:94-106. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  44. zur Nieden NI, Kempka G, Ahr HJ: In vitro differentiation of embryonic stem cells into mineralized osteoblasts.

    Differentiation 2003, 71:18-27. PubMed Abstract | Publisher Full Text OpenURL

  45. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T: Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.

    Cell 1997, 89:755-764. PubMed Abstract | Publisher Full Text OpenURL

  46. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ: Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.

    Cell 1997, 89:765-771. PubMed Abstract | Publisher Full Text OpenURL

  47. Feng H, Danfelter M, Strömqvist B, Heinegård D: Extracellular matrix in disc degeneration.

    J Bone Joint Surg Am 2006, 88(Suppl 2):25-29. PubMed Abstract | Publisher Full Text OpenURL

  48. Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Nöth U: Major biological obstacles for persistent cell-based regeneration of articular cartilage.

    Arthritis Res Ther 2007, 9:213-228. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  49. Pelttari K, Steck E, Richter W: The use of mesenchymal stem cells for chondrogenesis.

    Injury 2008, 39(Suppl 1):58-65. OpenURL

  50. Kwun IS, Cho YE, Lomeda RA, Shin HI, Choi JY, Kang YH, Beattie JH: Zinc deficiency suppresses matrix mineralization and retards osteogenesis transiently with catch-up possibly through Runx 2 modulation.

    Bone 2010, 46:732-741. PubMed Abstract | Publisher Full Text OpenURL

  51. Fromigué O, Marie PJ, Lomri A: Bone morphogenetic protein-2 and transforming growth factor-beta2 interact to modulate human bone marrow stromal cell proliferation and differentiation.

    J Cell Biochem 1998, 68:411-426. PubMed Abstract | Publisher Full Text OpenURL

  52. Bosetti M, Boccafoschi F, Leigheb M, Bianchi AE, Cannas M: Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering.

    J Tissue Eng Regen Med 2012, 6:205-213. PubMed Abstract | Publisher Full Text OpenURL