B. Hoffmann

Holger Jahr

M. Tingart

Mersedeh Tohidnezhad

S. Schneider

T. Nellesen

T. Pufe

U. Schnakenberg

Journal of Orthopaedic Translation

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Effects of platelet-released growth factors and monoaxial strain on tenocytes in vitro

Tohidnezhad, Mersedeh

Schneider, S.

Nellesen, T.

Hoffmann, B.

Schnakenberg, U.

Tingart, M.

Pufe, T.

Jahr, Holger

Elsevier - Publisher Connector 

110 Session: Regenerative Medicinecollagen expression through ALK5 in these cells. Our findings suggest that hypoxic
culture conditions for cartilage tissue engineering may benefit from an auto-regu-
latory cytokine signalling loop.
http://dx.doi.org/10.1016/j.jot.2016.06.197TGFb SUPERFAMILY-DEPENDENT AND OSMOLARITY-MEDIATED TYPE II
COLLAGEN EXPRESSION BY CHONDROCYTES IN VITRO
Ufuk T. Timur a,b, M. Caron a, A. van der Windt c, P. Emans a, T. Welting a,
Holger Jahr a,b
aDepartment of Orthopaedic Surgery, Maastricht University Medical Centre,
Maastricht, The Netherlands
bDepartment of Orthopaedics, University Hospital RWTH Aachen, Aachen,
Germany
cDepartment of Orthopaedics, Erasmus MC University Medical Centre, Rotterdam,
The Netherlands
Background: In vitro expansion of human articular chondrocytes (HACs) is required
for cell-based therapies to treat cartilage pathologies, because cartilage has a
poor intrinsic repair capacity. During standard expansion culture (i.e., plasma os-
molarity, 280 mOsm/kg) chondrocytes inevitably lose their specific phenotype and
de-differentiate. Unfortunately, this makes them inappropriate for autologous
chondrocyte implantation, or related, chondral repair techniques. It has been
shown that slightly elevated, but for articular chondrocytes physiological, osmolar-
ity (i.e., 380 mOsm/kg) increases type II collagen (COL2) expression in vitro. The
underlying molecular mechanism is, however, currently unknown. The transform-
ing growth factor beta (TGFb) superfamily members are accepted key regulators of
chondrocyte differentiation and further well-known to stimulate COL2 synthesis in
a variety of cell types, among which are chondrocytes. In this study, we aimed to
elucidate the contribution of the TGFb superfamily signalling as a putative molec-
ular mechanism that potentially stimulates COL2 expression under physiological
hyperosmolarity in vitro.
Subjects and Methods: HACs(P1) from patients undergoing knee arthroscopy were
cultured in cytokine-free medium of 280 or 380 mOsm/kg, under standard 2D in vi-
tro conditions, with or without lentiviral TGFb2 RNA interference (RNAi, Sigma-
Aldrich shRNA library). Expression of TGFb isoforms, BMPs, and chondrocyte
marker genes was evaluated by real-time qRT-PCR. TGFb2 protein secretion was
evaluated using ELISA and bioactivity was determined using an established re-
porter cell line. Involvement of BMP signalling was further investigated by
culturing HACs in the presence or absence of dorsomorphin (10 mM), an established
pharmacological inhibitor of these cytokines.
Results: Physiological osmolarity increased TGFb2 and TGFb3 mRNA expression,
TGFb2 protein secretion, as well as general TGFb activity under 380 mOsm/kg.Figure 1 Relative normalised COL2, BMP2, BMP4 and BMP6 mRNA expression in 280 (
(KD). Expression ratios of 280 mOsm control condition without virus are set to 1. DifferUpon TGFb2 isoform-specific shRNA-mediated knockdown, COL2 mRNA expression
was induced (Fig. 1). TGFb2 RNAi further induced expression of several BMPs to
large divergent extents (Fig. 1, data not shown), which were further up-regulated
under physiological osmolarity (Fig. 1). Physiological osmolarity-induced COL2
mRNA expression was, in contrast, secondarily suppressed by BMP-inhibitor dorso-
morphin (data not shown).
Discussion and Conclusion: TGFb2 knockdown under 380 mOsm increases COL2
expression in human chondrocytes in vitro, most likely through feedback regula-
tion of BMP signalling. BMP signalling itself is induced by osmolarity and increases
COL2 expression. Future studies will elucidate the BMP-mediated regulatory feed-
back loop upon TGF-b2 knockdown in more detail, as well as its influence on COL2
expression.
http://dx.doi.org/10.1016/j.jot.2016.06.198EFFECTS OF PLATELET-RELEASED GROWTH FACTORS AND MONOAXIAL STRAIN
ON TENOCYTES IN VITRO
Mersedeh Tohidnezhad a, S. Schneider a, T. Nellesen a, B. Hoffmann b,
U. Schnakenberg c, M. Tingart d, T. Pufe a, Holger Jahr d,e
aDepartment of Anatomy and Cell Biology, University Hospital RWTH Aachen,
Aachen, Germany
bForschungszentrumJu¨lich, Institute of Complex System (ICS-7) - Biomechanics,
Ju¨lich, Germany
cInstitute IWE-1, RWTH Aachen University, Aachen, Germany
dDepartment of Orthopaedics, University Hospital RWTH Aachen, Aachen, Germany
eDepartment of Orthopaedic Surgery, Maastricht University Medical Center,
Maastricht, The Netherlands
Background: Ruptured tendons heal very slowly and complete recovery from
injury is uncertain. Hypo-cellularity and poor nutrient supply through a lack of
vascularity make tissue regeneration challenging. In addition, tenocytes tend to
de-differentiate during routine in vitro culture, while tendon-derived progenitor
cells (TDCs) can differentiate into mature tenocytes under a variety of physiolog-
ical stimulations. As tendons naturally transmit loads between muscles and bones,
they withstand substantial levels of strain in situ. Tensile strain is thus, an impor-
tant environmental factor for tendon homeostasis from which regenerative in vitro
approaches may benefit. However, current evidence is ambiguous and cyclic ten-
sile strains of different frequencies and amplitudes appear to exert different ef-
fects, ranging from promoting tenogenic differentiation, to inducing non-
tenocyte lineage differentiation. Platelet-rich plasma (PRP) is currently being
widely clinically tested as a soft tissue-healing agent and may accelerate tendon
repair. More specifically, platelet-released growth factors (PRGF) may promote
TDCs differentiation and stabilise their phenotype in vitro. Here, we used a novelwhite) and 380 (black) mOsm cultures and upon TGF-b isoform-specific knockdown
ences from control condition are indicated by ** (p<0.005).
Figure 1 Strain- and PRGF-dependency of tenocyte marker gene expression. Shown are fluorescent intensities of Scx (A) and Tnmd (B) specific signals six hours post
treatment and respective IF signals of Scx (C) and Tnmd (D) at 24 hours. Static controls are shown in black, stretched conditions in grey. (mVEGF) in response to strain
amplitude (% relative elongation) and frequency (Hz). ** p <0.05; *** p < 0.01.
Session: Regenerative Medicine 111bioreactor to characterize TDC differentiation under the synergistic influence of
strain and PRGF.
Subjects and Methods: Neonatal murine Achilles’ tenocytes were isolated and
seeded into a custom-made bioreactor system at 25,000 cells/cm2, directly onto
custom collagen-coated (1 mg/ml) silicone membranes (polydimethylsiloxane,
PDMS; base: cross-linker ratioZ 1:40). Physiological levels of uniaxial cyclic strain
were assumed to fall within frequencies between 0.5e1.0 Hz and amplitudes of
5e10% relative stretching. PDGF was obtained from platelet concentrates (PC)
within a day after platelet apheresis in accordance with local ethical approval
(EK116/10, RWTH Aachen University). PC was washed twice with sodium citrate
buffer prior to collection at 2,000g for 10 minutes. Thrombocytes were concen-
trated two-fold and activated using bovine thrombin (10 U/ml; Sigma, Germany)
in 2.5 mM CaCl2. Real-time qRT-PCR was used to study the gene expression level
of tenogenic markers scleraxis (Scx) and tenomodulin (Tnmd) 6 and 24 hrs postFigure 2 Strain-dependency of VEGF expression. Time-dependent release of mu-
rine vascular endothelial growth factor (mVEGF) in response to strain amplitude (%
relative elongation) and frequency (Hz). ** p <0.05; *** p < 0.01.stimulation. Commercially available ELISA kits were used to quantify stretch-medi-
ated growth factor release; while immunofluorescent (IF) signal intensities were
quantified using Image J. Statistics was performed using GraphPad Prism software.
Results: Six hours after stimulation, both strain and PDGF, stimulated Scx expres-
sion (Fig. 1A), with the largest induction observed with low, rather than high, con-
centrations of PRGF (1% and 5%, respectively). Here, combining strain and PDGF
resulted in synergistic effects that only marginally increased further in time for
1% PRGF (Fig. 1B). Interestingly, there was a trend towards catch up-expression
of Scx in the static control upon stimulation with 1% PRGF. In contrast, Tnmd
expression was largely unresponsive to PRGF stimulation within six hours post
treatment (Fig. 1B), while strain application significantly increased its early
expression (Fig. 1B). After 24 hours, however, only 1% PRGF stimulated Tnmd
expression, while 5% had a suppressive effect. Without PRGF stimulation, stretch-
ing was able to stimulate Tnmd expression (Fig. 1D). Additional stretching only
marginally increased Tnmd expression any further (Fig. 1D, 1%). Surprisingly, 5%
PRGF appeared to suppress Tnmd expression under static conditions. Expression
of VEGF, a strong pro-angiogenic cytokine, in response to strain was studied at 6
and 24 hours post TDC stimulation, respectively (Fig. 2). The amplitude of stretch
rather than its frequency correlated positively with its expression: while 5% of
stretching revealed a strong induction of VEGF secretion at both time points,
VEGF secretion was stimulated to about the same extent independent of the fre-
quency (0.5 or 1 Hz, respectively) and increased in time (Fig. 2). In contrast,
doubling the strain amplitude to 10% reduced secretion levels of this cytokine,
also rather independent of its frequency. However, at this amplitude, 0.5 Hz
appeared to have the biggest stimulatory effect on VEGF.
Discussion and Conclusion: When aiming at stimulating VEGF expression, TDC cul-
ture at low amplitude stretching between 0.5e1 Hz appears beneficial. Histopath-
ological studies of end-stage symptomatic Achilles’ tendons revealed, among
others, an increased vascularity and VEGF is thus, negatively associated with dis-
ease progression. In contrast, during the regenerative phase, attracting vascula-
ture may be beneficial. With respect to tenocyte markers Scx and Tnmd, 1% of
PRGF can compensate to quite some extent for mechanical stimulation at a longer
term. Further experiments are also addressing extracellular matrix components as
differentiation markers. Our work provides interesting insights into TDC pheno-
typic stability in a strain- and PRGF-induced environment and supports the thera-
peutic potential of TDCs.
http://dx.doi.org/10.1016/j.jot.2016.06.205


Effects of platelet-released growth factors and monoaxial strain on tenocytes in vitro 

Effects of platelet-released growth factors and monoaxial strain on tenocytes in vitro

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