Biorheology 46 (2009) 45–55 45

DOI 10.3233/BIR-2009-0520
IOS Press

Effect of TGF β1, BMP-2 and hydraulic

pressure on chondrogenic differentiation of
bovine bone marrow mesenchymal
stromal cells
Stephan Zeiter a,∗ , Patrick Lezuo a and Keita Ito a,b
a
AO Research Institute, Davos, Switzerland
b
Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands

Received 18 September 2008

Revised 21 November 2008

Abstract. Bioactive factors, such as TGF β and BMP-2, as well as mechanical factors i.e. compressive loading and hydraulic
pressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells
(BMSCs). Since these factors are intracellularly transduced through different mechanisms, it is hypothesized that TGF β,
BMP-2 and hydraulic pressure may act synergistically on chondrogenic differentiation of BMSCs. Aggregates of bovine BMSC
were cultured in the presence of 10 ng/ml TGF β1 , 50 ng/ml BMP-2 or both. Half of the samples were loaded for 4 hours per
day with 0.5–3 MPa cyclic hydraulic pressure at 1 Hz. After 14 days of culture/loading, gene expression of chondrogenic
genes was assessed. DNA as well as glycosaminoglycan (GAG) content of the pellets were analysed. Neither pressure nor
BMP-2 had an influence on GAG/DNA content. However, cells responded to the presence of TGF β1 with an up-regulation of
chondrogenic genes and GAG/DNA of the aggregates increased compared to controls demonstrating the cells ability to respond
to external stimuli. The used concentrations of BMP-2 and parameters for pressure were neither able to induce nor modulate
chondrogenesis of bovine BMSCs and thus no synergistic effects were observed.
Keywords: Chondrogenesis, mechanobiology, bone marrow, stem cell, growth factors

1. Introduction

Intervertebral disc degeneration is a prevalent disease associated with high morbidity, which can re-
sult in disc herniation, spinal stenosis, radiculopathy and instability. Current treatments such as disc
prostheses, spinal fusion or conservative therapy often provide pain relief but are not without their own
problems [1,4,16,17,42,43]. The functional capacity of the intervertebral disc (IVD) is derived from the
material properties of its tissue which is composed of mainly protoeglycans (PG) and collagen type II
[8]. Its self repair and/or regenerative capacities are limited due to its low cell density and lack of vas-
cularisation. Bone marrow derived mesenchymal stromal cells (BMSCs) represent an attractive easily
available cell source for cell-based biological treatments [7,14,19,29]. To fulfil this challenge, BMSCs
*
Address for correspondence: Stephan Zeiter, AO Research Institute, Clavadelerstrasse 8, 7270 Davos Platz, Switzerland.
Tel.: +41 81 414 23 11; Fax: +41 81 414 22 88; E-mail: stephan.zeiter@aofoundation.org.

0006-355X/09/$17.00 © 2009 – IOS Press and the authors. All rights reserved
46 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis

have two outstanding properties: high proliferation capacity and the ability to differentiate into a chon-
drogenic lineage [31]. In a series of animal experiments, Sakai et al. demonstrated partial restoration of
the disc height and re-establishing of a PG containing extracellular matrix (ECM) after transplantation
of BMSCs into degenerative IVDs of rabbits. In addition the authors suggest that BMSC survive, pro-
liferate and differentiate into chondrocyte-like phenotype after transplantation [34,35]. Hence, BMSC
transfer could overcome the degenerative process to some extend in a small animal model.
To enhance the full potential of the BMSC treatment, preconditioning may be considered, particularly
since the IVD is a harsh environment which is acidic, hypoxic and poor in nutrients. The diffusion of
nutrients could be the limiting factor especially in human or large animal IVDs. Preconditioning towards
a chondrogenic phenotype of the BMSCs prior to transplantation might help to maintain cell viability
under these unfavourable conditions. Furthermore, since the function of the IVD is highly dependent on
its ECM, preconditioning of transferred cells towards a chondrogenic phenotype will increase production
of collagen type II and PG.
In vitro, TGF β is most often used to induce differentiation of BMSCs into chondrogenic cells [13,21,
31,44]. But other growth factors such as BMP-2, -4 and -6 have also been shown to have an influence on
chondrogenesis of these cells [37–39]. More recently the mechanical environment has been identified
as an important factor. It was demonstrated that cyclic hydraulic pressure modulates chondrogenic dif-
ferentiation of BMSCs induced by TGF β [2,3,24,25,40]. In experiments by Huang et al. [11,12] cyclic
compressive loading promoted chondrogenic differentiation of rabbit BMSCs in the absence of TGF β
emphasising the inductive role of the mechanical environment.
TGF β, BMP-2 and mechanical factors influence chondrogenic differentiation of BMSCs via different
mechanisms. TGF β and BMP-2 belong both to the TGF β superfamily of ligands. These ligands bind
to a type II receptor which phosphorylates a type I receptor. Type I receptor phoshorylates receptor-
regulated SMAD (R-SMAD) which then bind to common-partner SMAD 4 (co-SMAD 4). Finally these
complexes act as transcription factors in the nucleus. Although TGF β and BMP-2 belong to the same su-
perfamily of ligands, they bind to different receptors (TGF β receptor and BMP-2 receptor, respectively)
which lead to activation of different R-SMAD molecules: SMAD 2 and 3 are TGF β receptor-associated
SMAD and SMAD 1, 5 and 8 are BMP-associated SMAD. All activated SMAD bind to the co-SMAD 4
[23,26]. Binding of either ligand can result in an up-regulation of so-called chondrogenic genes Sox-9,
collagen type II and aggrecan. For the mechanical factors, Huang et al. demonstrated that cyclic com-
pressive loading leads to up-regulation of endogenous TGF β gene expression as well as upregulation
of type I and type II and TGF β receptors. Hence, TGF β signal transduction seems to be involved in
chondrogenesis of BMSCs induced by compressive loading [11,12]. In summary, bioactive factors and
mechanical factors act through similar but different pathways to influence chondrogenesis of BMSCs.
However, little is known about the combined effect of TGF β, BMP-2 and mechanical loading on
differentiation of BMSCs. This study investigates the influence of all three factors on chondrogenesis
of BMSCs on their own, or in combination with each other. It is hypothesized that TGF β, BMP-2 and
hydraulic pressure act synergistically to enhance chondrogenic differentiation of BMSC.

2. Materials and methods

2.1. Cell harvest, cell culture and pellet formation

Bone marrow was harvested from the pelvises of three calves (4 months old) within half an hour
after slaughtering. Aspirates were immediately mixed with 5000 IU Heparin (Fresenius Medical Care,
 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis 47

Switzerland) to inhibit clotting. In the laboratory, bone marrow was washed with Tyrrode’s Balanced Salt
Solution and centrifuged at 400g for 8 min. After removal of the supernatant, cells were then resuspended
in Minimum Essential Medium alpha supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin,
20 mM HEPES buffer solution and 10% fetal bovine serum (all Gibco, Paisley, Scotland). Finally, cells
from approximately 5 ml bone marrow aspirate were plated into 300 cm2 plastic culture flasks (TPP,
Trasadingen, Switzerland). Medium was changed twice a week. Adherent cells were expanded up to
passage 3 (up to 15 days of culture). Cells were sub-cultured at approximately 80% confluency. At each
passage, cells were trypsinized and washed in serum containing medium. Cells were then reseeded at a
density of 5000 cells/cm2 .
For the experiments, aliquots of 0.2 × 106 cells were suspended in 2 ml serum free basic medium con-
sisting of high glucose (4500 mg/ml) Dulbecco’s Modified Eagle Medium supplemented with 100 U/ml
penicillin, 100 µg/ml streptomycin, 20 mM HEPES buffer solution, 1% non-essential amino acids
(Gibco, Paisley, Scotland) and 1% ITS+ (Sigma, Steinheim, Germany; final concentration: 1.0 mg/ml in-
sulin from bovine pancreas, 0.55 mg/ml human transferrin, 0.5 µg/ml sodium selenite, 50 mg/ml bovine
serum albumin and 470 µg/ml linoleic acid), 10−7 M dexamethasone and 50 µg/ml ascorbic acid 2
phosphate (both Sigma, Steinheim, Germany). Cell suspensions were transferred into sterile 15 ml tubes
(TPP, Trasadingen, Switzerland) and cells were centrifuged at 500g for 5 min. After 48 h the supernatant
was carefully removed and the formed cell pellet was placed into standard 1.5 ml polypropylene tubes
(Eppendorf AG, Hamburg, Germany) in which the lid was cut open (1 pellet per tube). These tubes were
then sealed using a 0.254 mm thick gas permeable medical grade silicone membrane (Nusil Technology,
Carpinteria, CA, USA) carefully avoiding any air bubbles. Proper sealing was assured by a rubber ring
(Sahli AG, Switzerland). The flexible membrane allowed transmission of hydraulic pressure to the cell
pellet.

2.2. Application of hydraulic pressure

To apply hydraulic pressure, half of the samples were placed into a custom-made stainless steel pres-
sure vessel filled with pre-warmed (37◦ C) sterilized distilled water. The vessel was situated inside an
incubator set at 5% CO2 , 21% O2 , 37◦ C and 90% humidity. A computer-controlled water hydraulic ram
driven by a pneumatic actuator (SP1250, Enduratec, Eden Prairie, MN, USA) generated intermittent
sinusoidal hydraulic pressure at a frequency of 1 Hz. A temperature compensated pressure transducer
(Keller AG, Winterthur, Switzerland) inside the pressure vessel provided the feedback signal for PID
control of the actuator. Samples were loaded with 0.5–3 MPa intermittent cyclic sinusoidal hydraulic
pressure at 1 Hz for 4 h each day starting the day pellets were transferred into the modified 1.5 ml tubes.
When not loaded, the tubes containing the pellets were taken out of the pressure vessel and air dried
under a laminar flow hood for 20 min before placing back into the incubator (same settings as before)
until the next loading session.

2.3. Experimental design

Depending on the stimulation group, media consisted either of serum free basic media (see above)
or basic media with addition of either 10 ng/ml TGF β1 , 50 ng/ml BMP-2 or both. Half of the samples
were exposed to hydraulic pressure. Hence, the experiment consisted of eight groups (control, hydraulic
pressure only and TGF β1 , BMP-2 or both, all with or without loading). Unloaded samples were prepared
and maintained under identical conditions, but not placed into the pressure vessel. The medium was
changed every third day prior to the application of hydraulic pressure.
48 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis

2.4. Pellet harvesting and biochemical analysis

Pellets were harvested before the first loading session (control day 1) and after 14 days of culture.
Loaded samples were collected within half an hour after loading. After careful removal of the medium,
pellets were either digested with 0.5 ml proteinase K (0.5 mg/ml; Roche, Basel, Switzerland) for 15–18 h
at 56◦ C for determination of DNA and total sulphated glycosaminoglycan (GAG) content or placed in
1 ml TRI reagent (Molecular Research Center, Cincinnati, OH, USA) supplemented with 6 µl poly-
acrylcarrier (Molecular Research Center, Cincinnati, OH, USA). Subsequently, samples were frozen at
−20◦ C until further analysis. Samples were thawed at room temperature and DNA content was measured
spectrofluorometrically using Hoechst 33258 dye (Polysciences Inc., Warrington, PA, USA) and puri-
fied calf thymus DNA as standard [15]. DNA data was analysed relative to day 1. The amount of GAG
was determined by the dimethylmethylene blue (DMMB) dye binding method, using bovine chondroitin
sulfate as standard [9]. GAG content of pellets was normalized to DNA content.

2.5. Gene expression analysis

Total cellular RNA was isolated using a modified TRIspin method [32]. Briefly, 1-bromo-3-
chloropropane (0.1 ml per 1 ml TRI reagent) was added and centrifuged. The resulting aqueous phase
containing the RNA was transferred to a fresh column of the GenElute™ Mammalian Total RNA
Miniprep Kit (Sigma, Steinheim, Germany) and RNA was isolated according to the manufactures pro-
tocol. Resulting RNA was reverse transcribed to cDNA with TaqMan Reverse Transcription Reagents
(Applied Biosystems, Foster City, CA, USA). Finally, real time polymerase chain reaction (PCR) was
performed on a 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA) under stan-
dard thermal conditions using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City,
CA, USA) and gene specific primers and probes for Sox-9, collagen type I, collagen type II and aggrecan.
These oligonucleotide primers and TaqMan probes (Table 1; all from Microsynth, Balgach, Switzerland)
were designed with Primer Express Oligo Design software, versions 1.5/2.0 (Applied Biosystems, Foster
City, CA, USA). Probes were labelled with the reporter dye molecule FAM (6-carboxyfluorescein) at the
5 -end and with the quencher dye TAMRA (6-carbox-N,N,N ,N -tetramethylrhodamine) at the 3 -end.
To exclude amplification of genomic DNA, the probe or one of the primers were selected to overlap

Table 1
Sequences of primers and probes used for real time PCR
Gene Primer fw 5 –3 Primer rev 5 –3 Probe 5 –3
Sox-9 AAC GCC GAG ACG AAC GGC TTC AGC AGT CTC
CTC AGC AAG CGC TTC TC CAG AGC TTG CCC A

Aggrecan CCA ACG AAA CCT GCA CTC GTT ATG TTG CAT AGA
ATG ACG TGT ACT GGC TGC CTC AGA CCT CGC CCT CCA
Procollagen 1A2 TGC AGT AAC TTC CGC GTG GTC CAT GCC AAT CCT TAC
GTG CCT AGC A CTC TAT CTC CA AAG AGG CAA CTG C
Procollagen 2A1 AAG AAA CAC ATC TGG GAG CCA CAA CGG TGG CTT
TGG TTT GGA GAA A GGT TGT CAT C CCA CTT CAG CTA TGG
 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis 49

an exon–exon junction. For PCR analysis 18S ribosomal RNA (endogenous control) TaqMan Gene
Expression Assays (both from Applied Biosystems, Foster City, CA, USA) was used. TaqMan Gene
Expression Assays are pre-designed gene-specific probe and primer sets. Relative quantification of tar-
get mRNA was performed according to the comparative CT method [20] with 18S ribosomal RNA as
endogenous control, since it has been shown to be the most appropriate endogenous control for studies
on mechanobiology of cartilaginous tissues [18], and control day 1 as reference. Hence, gene expres-
sion levels of cells harvested at day 14 were normalized to the control values at day 1 of the respective
genes.

2.6. Statistics

In this study three independent factors (TGF β1 , BMP-2 and hydraulic pressure) and their combina-
tion (eight different experimental groups) were investigated. Statistical analysis for difference between
groups was not done due to the large number of possible comparisons relative to the sample sizes. How-
ever, statistical analysis for an effect of each treatment was carried out by reducing the comparisons to
only that at day 1. This was done by Kruskal–Wallis test for differences among groups and subsequent
one-tailed Mann–Whitney post-hoc testing (p  0.05).

3. Results

3.1. Biochemical analysis

DNA content relative to day 1 decreased significantly after 14 days in culture except for the group
cultured in the presence of TGF β1 only. Loaded specimens were not different from their unloaded
counterparts with the same growth factors. The amount of DNA of the control pellets at day 14 dropped
to 71% pellets compared to day 1. Loaded samples cultured without growth factors had a decrease
to 72%. Pellets cultured in the presence of BMP-2 were not much different with 75 and 73%, with-
out and with loading, respectively. In the presence of TGF β1 DNA content was almost unchanged
(90% and 93%, with and without loading, respectively). Both growth factors combined resulted in
similar DNA contents to TGF β1 alone (87% and 83%, with and without loading, respectively) (see
Fig. 1).
Small amounts of GAGs were detected at day 1 (0.99 ng GAG/ng DNA). Control and BMP had
fewer GAGs per DNA after 14 days of culture regardless if samples were loaded or not. BMP-2 slightly
increased GAG content of the pellets compared to control (0.59 and 0.82 ng GAG/ng DNA, respec-
tively). The addition of TGF β1 was the only factor that significantly increased GAG per DNA con-
tent of the pellets. A higher GAG/DNA content was found in samples cultured in the presence of
TGF β1 (4.54 ng GAG/ng DNA) or TGF β1 and BMP-2 (4.95 ng GAG/ng DNA). If, in addition,
it was combined with hydraulic pressure, the GAG content per DNA did not increase further (see
Fig. 2).

3.2. Gene expression analysis

BMSCs gene expression levels were affected by treatment. TGF β1 alone or in combination with
BMP-2 lead to a significant up-regulation of the chondrogenic genes collagen type II and aggrecan
50 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis

Fig. 1. DNA content of bovine BMSCs pellet cultures at day 14 relative to day 1 controls; white boxes represent unloaded
samples, grey boxes loaded samples; “*” indicates a significant difference to day 1; n = 3 animals.

Fig. 2. GAG/DNA content of bovine MSC cultured in pellets at day 1 and 14; white boxes represent unloaded samples, grey
boxes loaded samples; “*” indicates a significant difference to day 1; n = 3 animals.

compared to day 1. BMP-2 alone also significantly upregulated these genes, however to a smaller extend
than TGF β1 . Collagen type I was increased in the presence of TGF β1 , but not by loading or BMP-2
alone. TGF β1 alone significantly upregulated Sox-9 expression. Overall, there was no to little effect
of loading compared to the corresponding growth factor unloaded control, except for Sox-9, where
additional loading lead to downregulation, especially in combination with BMP-2 (see Fig. 3).
 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis 51

Fig. 3. Effect of growth factors BMP-2 and TGF β1 and hydraulic pressure (0.5–3 MPa, 1 Hz, 4 h/day) on gene expression
of collagen type I (coll I), Sox-9, collagen type II (coll II) and aggrecan of bovine MSC cultured in pellets at day 14 relative
to control day 1; white boxes represent unloaded samples, grey boxes loaded samples; “*” indicates a significant difference to
day 1; logarithmic scale; n = 3 animals.

4. Discussion

Bioactive factors TGF β and BMP-2 as well as pressure have been shown to influence chondrogenesis
of BMSCs. This study aimed to investigate the synergistic potential of these factors during this process.
The BMSC pellet culture system mimics pre-cartilage condensation during embryonic development and
allows cell to cell interaction. Since chondrogenesis requires cell to cell interaction in a 3D environment,
the pellet culture system is a popular model for studying chondrogenesis in vitro. The small decrease in
DNA content over the early observation period is a common observation with BMSCs in pellet culture
and has been described previously by Yoo et al. [44]. Since the BMSCs prepared with adhesion selection
are a heterogeneous cell population, it can be speculated that a subpopulation of cells cannot cope with
the 3D environment. Nevertheless, a majority of the cells continued to survive during the observation
period in the control and TGF β1 groups. Although some have observed a tendancy for bovine BMSCs
toward chondrogenesis without the addition of any bioactive factors [5], others [27] as well as in this
study have not observed such behaviour in their control groups. Finally, it was shown by Johnstone et
al. [13] that dexamethasone is essential to induce chondrogenesis in combination with TGF β. Since
52 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis

the goal of this study was to investigate the effect of TGF β, BMP-2 and hydraulic pressure each and
in combination with each other, it was necessary to use dexamethasone for the TGF β and hence for
all others to keep the basic media as uniform as possible. It is possible that dexamethasone can cause
differentiation of BMSCs, but our negative control (day 14) did not exhibit such behavior. Nevertheless,
TGF β is the factor most often used to induce chondrogenesis in BMSCs and can be regarded as a gold
standard or positive control. Thus, upregulation of chondrogenic genes and production of ECM confirm
that cells within our system were able to respond to an external stimulus, in agreement with the literature
[13,21,31,44], and that the used culture system consisting of modified tubes with the lid cut open, but
sealed with a gas permeable membrane is valid. This avoids the use of sealing bags which is cumbersome
and prone to contamination.
BMP-2 alone only had a minor effect on GAG production despite upregulation of the chondrogenic
genes collagen type II and aggrecan. In contrast to TGF β for which most often a concentration of
10 ng/ml is used to induce chondrogenesis, the concentration used for BMP-2 varies between 50 and
500 ng/ml [22,30,37–39,41]. A concentration of 50 ng/ml BMP-2 has been shown to be effective for hu-
man MSC isolated from the bone marrow [37] and fat tissue [22] and for bovine chondrogenic progenitor
cells isolated from the synovium [30]. In the latter study, after one week upregulation of Sox-9 reached
a plateau when a concentration of 50 ng/ml was used. Collagen type II and aggrecan were only slightly
more upregulated with 200 ng/ml BMP-2. Using the same concentrations (100 ng/ml) different effects of
BMP-2 have been reported after 14 days of culture: either no change in GAG content of the pellet [39] or
an increase [41] was noted. However, after 21 days in both studies GAG content had increased. Hence,
the observation period of this study might have been too short to detect an increase in GAG content of
the samples cultured in the presence of BMP-2. The effect of growth factors is not only dependent on
growth factor concentration and duration of exposure, but also on cell type, cell source, culture system
and species of donor. Chondrogenesis in human and rabbit MSCs isolated from the bone marrow or fat
tissue can be induced by TGF β or BMP-2 [22,37–39,41]. Bovine synovium derived progenitor cells
only respond to BMP-2, but not to TGF β [30] whereas bovine BMSCs in this study behave the opposite
way around. Therefore, the influence of cell type and species on chondrogenesis are most likely key
factors to be addressed in other studies.
For this study hydraulic pressure was chosen since the gelatinous center of the IVD is loaded in this
manner [28]. However, hydraulic pressure did not influence the DNA content or chondrogenesis of the
pellets compared to unloaded counterparts. In the literature, controversial results can be found for hy-
draulic pressure: although several authors have demonstrated an effect of hydraulic pressure on BMSCs
with or without TGF β [2,3,24,25,40], in other studies, such as this one, hydraulic pressure did not in-
fluence gene expression of aggrecan and collagen type II [10,36]. The effect of hydraulic pressure on
TGF β3 induced chondrogenesis of BMSCs has been shown to be time and amplitude dependent [24].
14 days of stimulation increased GAG/DNA as well as expression of chondrogenic genes significantly.
Although cells responded maximally to 10 MPa hydraulic pressure, even with 1 MPa, an increase of PG
was observed. We chose 3 MPa to better mimic physiological pressure levels within the IV disc. How-
ever, the downregulation of Sox-9 in loaded samples does indicate that the chosen loading regime was
not stimulatory. In future experiments different parameters (e.g. different pressure amplitudes, frequen-
cies and/or time of stimulation) should be explored. Since there was no modulation of TGF β induced
chondrogenesis, it is not surprising that hydraulic pressure on its own did not cause induction of this
process. Several studies have shown only limited to no induction of chondrogenesis, if hydraulic pres-
sure only was used [2,3,10,24,25,36]. On the other hand, compressive loading lead to an upregulation
of chondrogenic genes in BMSCs even in the absence of TGF β [6,11,12,27]. This may indicate that
 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis 53

BMSCs are sensitive to other components of compressive loading other than hydraulic pressure, e.g. de-
formation. Huang [11,12] and Mouw [27] have demonstrated that the TGF β pathway could be involved
in mechanotransduction of compressive loading. Unfortunately, to our knowledge the mechanotrans-
duction of hydrostatic pressure in BMSCs has not been investigated yet. Differences in how mechanical
stimulus is transduced might explain the differences between studies. However, at the moment it is too
early to draw any conclusion since available data is too heterogeneous between studies (e.g. species,
loading parameters, culture system). Further studies with more homogenous study designs are needed to
investigate these factors more systematically.
The function of the IVD is dependent on its ECM which consists mainly of collagen type II and I as
well as PG expression of these ECM components. The addition of TGF β1 resulted in an upregulation
of collagen type II and aggrecan and production of GAGs whereas the exposure to BMP-2, hydraulic
pressure and combination thereof had limited to no effect. It might be that other, not investigated genes
changed their expression in this experiment in the presence of these factors. Especially genes involved
within the above presented pathways and/or catabolic genes (i.e. metalloproteinases) are interesting
targets for future studies. Another factor that will need to be addressed in future similar experiments
is the effect of hypoxia, since the IVD represents a hypoxic environment and it has been shown that
hypoxia does influence TGF β1 induced chondrogenesis [33,36].
In summary, TGF β1 was the only factor having substantial effect on chondrogenesis of BMSCs in
this study. Although this study could not support our hypothesis, it provides useful information on the
effect of growth factors and hydraulic pressure on chondrogenesis of BMSCs albeit in an uncommon
way, i.e. instead of reporting what did work, it provides information what did not work. It is our belief,
that such outcomes should be reported to the scientific community as well, since it may save the efforts
of others. However, more extensive and standardised experiments are needed to elucidate the effect and
mechanism of bioactive and mechanical factors on chondrogenesis of BMSCs, e.g. what is the difference
in mechanotransduction of compression compared to pressure in chondrogenesis of BMSCs.

References

[1] P.A. Anderson and J.P. Rouleau, Intervertebral disc arthroplasty, Spine 29 (2004), 2779–2786.
[2] P. Angele, D. Schumann, M. Angele, B. Kinner et al., Cyclic, mechanical compression enhances chondrogenesis of mes-
enchymal progenitor cells in tissue engineering scaffolds, Biorheology 41 (2004), 335–346.
[3] P. Angele, J.U. Yoo, C. Smith, J. Mansour et al., Cyclic hydrostatic pressure enhances the chondrogenic phenotype of
human mesenchymal progenitor cells differentiated in vitro, J. Orthop. Res. 21 (2003), 451–457.
[4] J.N. Awad and R. Moskovich, Lumbar disc herniations: surgical versus nonsurgical treatment, Clin. Orthop. Relat. Res.
443 (2006), 183–197.
[5] D. Bosnakovski, M. Mizuno, G. Kim, T. Ishiguro et al., Chondrogenic differentiation of bovine bone marrow mesenchymal
stem cells in pellet cultural system, Exp. Hematol. 32 (2004), 502–509.
[6] J.J. Campbell, D.A. Lee and D.L. Bader, Dynamic compressive strain influences chondrogenic gene expression in human
mesenchymal stem cells, Biorheology 43 (2006), 455–470.
[7] C. Evans, Potential biologic therapies for the intervertebral disc, J. Bone Joint Surg. Am. 88(Suppl. 2) (2006), 95–98.
[8] D.R. Eyre, Biochemistry of the intervertebral disc, Int. Rev. Connect. Tissue Res. 8 (1979), 227–291.
[9] R.W. Farndale, D.J. Buttle and A.J. Barrett, Improved quantitation and discrimination of sulphated glycosaminoglycans
by use of dimethylmethylene blue, Biochim. Biophys. Acta 883 (1986), 173–177.
[10] A.R. Finger, C.Y. Sargent, K.O. Dulaney, S.H. Bernacki and E.G. Loboa, Differential effects on messenger ribonucleic
acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and
steady applications of cyclic hydrostatic pressure, Tissue Eng. 13 (2007), 1151–1158.
[11] C.Y. Huang, K.L. Hagar, L.E. Frost, Y. Sun and H.S. Cheung, Effects of cyclic compressive loading on chondrogenesis of
rabbit bone-marrow derived mesenchymal stem cells, Stem Cells 22 (2004), 313–323.
54 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis

[12] C.Y. Huang, P.M. Reuben and H.S. Cheung, Temporal expression patterns and corresponding protein inductions of early
responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading, Stem Cells 23
(2005), 1113–1121.
[13] B. Johnstone, T.M. Hering, A.I. Caplan, V.M. Goldberg and J.U. Yoo, In vitro chondrogenesis of bone marrow-derived
mesenchymal progenitor cells, Exp. Cell Res. 238 (1998), 265–272.
[14] C. Jorgensen, J. Gordeladze and D. Noel, Tissue engineering through autologous mesenchymal stem cells, Curr. Opin.
Biotechnol. 15 (2004), 406–410.
[15] C. Labarca and K. Paigen, A simple, rapid, and sensitive DNA assay procedure, Anal. Biochem. 102 (1980), 344–352.
[16] C.K. Lee, Accelerated degeneration of the segment adjacent to a lumbar fusion, Spine 13 (1988), 375–377.
[17] C.K. Lee and N.A. Langrana, A review of spinal fusion for degenerative disc disease: need for alternative treatment
approach of disc arthroplasty?, Spine J. 4 (2004), 173S–176S.
[18] C.R. Lee, S. Grad, J.J. MacLean, J.C. Iatridis and M. Alini, Effect of mechanical loading on mRNa levels of common
endogenous controls in articular chondrocytes and intervertebral disk, Anal. Biochem. 341 (2005), 372–375.
[19] V.Y. Leung, D. Chan and K.M. Cheung, Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limi-
tations, and future direction, Eur. Spine J. 15 (2006), 406–413.
[20] K.J. Livak and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the
2(-Delta Delta C(T)) method, Methods 25 (2001), 402–408.
[21] A.M. Mackay, S.C. Beck, J.M. Murphy, F.P. Barry, C.O. Chichester and M.F. Pittenger, Chondrogenic differentiation of
cultured human mesenchymal stem cells from marrow, Tissue Eng. 4 (1998), 415–428.
[22] A.T. Mehlhorn, P. Niemeyer, K. Kaschte, L. Muller et al., Differential effects of BMP-2 and TGF-beta1 on chondrogenic
differentiation of adipose derived stem cells, Cell Prolif. 40 (2007), 809–823.
[23] A. Mehra and J.L. Wrana, TGF-beta and the Smad signal transduction pathway, Biochem. Cell Biol. 80 (2002), 605–622.
[24] K. Miyanishi, M.C. Trindade, D.P. Lindsey, G.S. Beaupre et al., Dose- and time-dependent effects of cyclic hydrostatic
pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro,
Tissue Eng. 12 (2006), 2253–2262.
[25] K. Miyanishi, M.C. Trindade, D.P. Lindsey, G.S. Beaupre et al., Effects of hydrostatic pressure and transforming growth
factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro, Tissue Eng. 12 (2006), 1419–1428.
[26] K. Miyazawa, M. Shinozaki, T. Hara, T. Furuya and K. Miyazono, Two major Smad pathways in TGF-beta superfamily
signalling, Genes Cells 7 (2002), 1191–1204.
[27] J.K. Mouw, J.T. Connelly, C.G. Wilson, K.E. Michael and M.E. Levenston, Dynamic compression regulates the expression
and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells, Stem Cells 25 (2007), 655–663.
[28] A. Nachemson, Disc pressure measurements, Spine 6 (1981), 93–97.
[29] G. Paesold, A.G. Nerlich and N. Boos, Biological treatment strategies for disc degeneration: potentials and shortcomings,
Eur. Spine J. 16 (2007), 447–468.
[30] Y. Park, M. Sugimoto, A. Watrin, M. Chiquet and E.B. Hunziker, BMP-2 induces the expression of chondrocyte-specific
genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel, Osteoarthr. Cartil. 13
(2005), 527–536.
[31] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal et al., Multilineage potential of adult human mesenchymal stem
cells, Science 284 (1999), 143–147.
[32] C. Reno, L. Marchuk, P. Sciore, C.B. Frank and D.A. Hart, Rapid isolation of total RNA from small samples of hypocel-
lular, dense connective tissues, BioTechniques 22 (1997), 1082–1086.
[33] M.V. Risbud, T.J. Albert, A. Guttapalli, E.J. Vresilovic et al., Differentiation of mesenchymal stem cells towards a nucleus
pulposus-like phenotype in vitro: implications for cell-based transplantation therapy, Spine 29 (2004), 2627–2632.
[34] D. Sakai, J. Mochida, T. Iwashina, T. Watanabe et al., Differentiation of mesenchymal stem cells transplanted to a rabbit
degenerative disc model: potential and limitations for stem cell therapy in disc regeneration, Spine 30 (2005), 2379–2387.
[35] D. Sakai, J. Mochida, Y. Yamamoto, T. Nomura et al., Transplantation of mesenchymal stem cells embedded in Atelo-
collagen((R)) gel to the intervertebral disc: a potential therapeutic model for disc degeneration, Biomaterials 24 (2003),
3531–3541.
[36] K. Scherer, M. Schunke, R. Sellckau, J. Hassenpflug and B. Kurz, The influence of oxygen and hydrostatic pressure on
articular chondrocytes and adherent bone marrow cells in vitro, Biorheology 41 (2004), 323–333.
[37] B. Schmitt, J. Ringe, T. Haupl, M. Notter et al., BMP2 initiates chondrogenic lineage development of adult human mes-
enchymal stem cells in high-density culture, Differentiation 71 (2003), 567–577.
[38] I. Sekiya, B.L. Larson, J.T. Vuoristo, R.L. Reger and D.J. Prockop, Comparison of effect of BMP-2, -4, and -6 on in vitro
cartilage formation of human adult stem cells from bone marrow stroma, Cell Tissue Res. 320 (2005), 269–276.
[39] W.S. Toh, H. Liu, B.C. Heng, A.J. Rufaihah, C.P. Ye and T. Cao, Combined effects of TGFbeta1 and BMP2 in serum-
free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation, Growth Factors 23
(2005), 313–321.
 S. Zeiter et al. / Effect of growth factors and pressure on chondrogenesis 55

[40] D.R. Wagner, D.P. Lindsey, K.W. Li, P. Tummala et al., Hydrostatic pressure enhances chondrogenic differentiation of
human bone marrow stromal cells in osteochondrogenic medium, Ann. Biomed. Eng. 36 (2008), 813–820.
[41] Y. Wei, Y. Hu, R. Lv and D. Li, Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the
use of rhBMP-2, Cytotherapy 8 (2006), 570–579.
[42] J.N. Weinstein, J.D. Lurie, T.D. Tosteson, J.S. Skinner et al., Surgical vs nonoperative treatment for lumbar disk herniation:
the Spine Patient Outcomes Research Trial (SPORT) observational cohort, JAMA 296 (2006), 2451–2459.
[43] J.N. Weinstein, T.D. Tosteson, J.D. Lurie, A.N. Tosteson et al., Surgical vs nonoperative treatment for lumbar disk herni-
ation: the Spine Patient Outcomes Research Trial (SPORT): a randomized trial, JAMA 296 (2006), 2441–2450.
[44] J.U. Yoo, T.S. Barthel, K. Nishimura, L. Solchaga et al., The chondrogenic potential of human bone-marrow-derived
mesenchymal progenitor cells, J. Bone Joint Surg. Am. 80 (1998), 1745–1757.