Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair.

Abstract

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin-like growth factor 1 (IGF-1; increases proteoglycan and collagen synthesis), beta-aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para-nitrophenyl-beta-D-xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF-1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used.

Full text

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Proteoglycan production is required in initial stages of new
cartilage matrix formation but inhibits integrative cartilage
repair


Journal: Journal of Tissue Engineering and Regenerative Medicine
Manuscript ID: TERM-08-0101.R1
Wiley - Manuscript type: Research Article
Date Submitted by the
Author:
10-Dec-2008
Complete List of Authors: Bastiaansen-Jenniskens, Yvonne; Erasmus MC University Medical
Center, Orthopaedics; TNO Quality of Life, Business Unit
BioSciences
Koevoet, Wendy; Erasmus MC, University Medical Centre,
Otorhinolaryngology
Feijt, Carola; Erasmus MC University Medical Center, Orthopaedics
Bos, Pieter; Erasmus MC University Medical Center, Orthopaedics
Verhaar, Jan; Erasmus MC University Medical Center, Orthopaedics
van Osch, Gerjo; Erasmus MC University Medical Center,
Orthopaedics; Erasmus MC, University Medical Center,
Otorhinolaryngology
DeGroot, Jeroen; TNO Quality of Life, Business Unit BioSciences
Keywords:
glycosaminoglycan, collagen, collagen cross-links, cartilage matrix,
chondrocyte, integrative repair




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YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 1/20
Proteoglycan production is required in initial stages of new cartilage matrix
formation but inhibits integrative cartilage repair

Y.M. Bastiaansen-Jenniskens1,2; W. Koevoet3; C. Feijt1; P.K. Bos1; J.A.N. Verhaar1;
G.J.V.M. VanOsch1,3; J. DeGroot2

1: Erasmus MC, University Medical Centre Rotterdam, dept. of Orthopaedics, the
Netherlands
2: TNO Quality of Life, Business Unit BioSciences, Leiden, the Netherlands
3: Erasmus MC, University Medical Centre Rotterdam, dept. of Otorhinolaryngology,
the Netherlands

Keywords: glycosaminoglycan, collagen, collagen cross-links, cartilage matrix,
chondrocyte, integrative repair

Supported by a grant of The Dutch Arthritis Association (Reumafonds), NR 02-2-40

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of proteoglycan formation with PNPX was most beneficial.
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Abstract
The optimal stimulus to repair or regenerate cartilage is not known. We therefore
modulated collagen deposition, collagen cross-linking and GAG deposition
simultaneously during cartilage matrix production and integrative repair, creating
more insight in their role in cartilage repair processes.
Insuline-like Growth Factor 1 (IGF-1, increases proteoglycan and collagen synthesis),
Beta-aminopropionitrile (BAPN, a reversible inhibitor of collagen crosslinking) and
para-nitrophenyl-beta-d-xyloside (PNPX, interferes with proteoglycan production)
were used. Bovine articular chondrocytes were cultured in alginate beads for three
weeks with or without IGF-1, BAPN or PNPX alone and in all possible combinations
followed by three weeks in control medium. DNA content, GAG and collagen
deposition and collagen cross-links were determined. Cartilage constructs were
cultured under same conditions and histologically analysed for integration of two
opposing cartilage matrices.
In alginate cultures, inhibition of collagen cross-linking with BAPN in combination with
promotion of matrix synthesis using IGF1 was most beneficial for matrix deposition.
Addition of PNPX was always detrimental for matrix deposition. For integration of
opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention
When a new matrix is produced, proteoglycans are important to retain collagen in the
matrix. When two already formed cartilage matrices have to integrate, temporary
absence of proteoglycans and temporary inhibition of collagen cross-linking might be
more beneficial in combination with stimulation of collagen production by for example
IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration
depends on the type of therapy that will be used.
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aminopropionitrile (BAPN). It was found that collagen cross-links are important for the
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1. Introduction
Once damaged, adult articular cartilage has a poor repair capacity, which is probably
due to the ineffective repair of the collagen network, since proteoglycan depletion is
often reversible (Lafeber 1992). Although collagen turnover is increased in osteoarthritis
(OA), this does not lead to the formation of a functional network (Aigner 1993;
Billinghurst 1997; Nelson 1998; Verzijl 2000). This suggests an activated repair
mechanism in OA that appears however ineffective in repairing or maintaining the
ECM homeostasis.
Several strategies are under investigation to promote matrix regeneration in cartilage
repair. Growth factors are used to modulate matrix production, by directly adding
them to chondrocytes in culture (Gooch 2002; Veilleux 2005) or by inducing over
expression of the growth factor of interest (Madry 2002; Kaul 2006). For example, we
and others found that addition of IGF1 to chondrocytes in culture resulted in more
proteoglycans and collagen than in the control condition without IGF1 (Blunk 2002;
Mauck 2003; De Mattei 2004; Jenniskens 2006)
In addition to growth factors, modulation of collagen network formation is also
employed to understand cartilage matrix formation and functionality, for example by
the inhibition of collagen cross-linking by inhibition of lysyl oxidase (LOX) with β-
integrative repair and adhesive strength of cartilage (DiMicco 2002) and that transient
inhibition of the formation of these cross-links improved integrative repair and
collagen cross-link maturation (McGowan 2005). In cultures of chondrocytes in
alginate, inhibition of collagen cross-link formation with BAPN resulted in an increase
of collagen production (Beekman 1997; Wong 2002; Bastiaansen-Jenniskens
2008a). In concordance with earlier explant studies (McGowan 2005), transient
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potentially complementary effects of these approaches, our hypothesis was that
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inhibition of LOX in alginate cultures resulted in accelerated cross-link maturation and
improved functionality of the newly formed matrix (Bastiaansen-Jenniskens 2008a).
The influence of glycosaminoglycans (GAGs) on cartilage growth and matrix
production is also under investigation, but gets less attention than collagen. GAG
depletion in cartilage explants resulted in less expansive growth and a more mature
matrix with increased tensile integrity (Asanbaeva 2007). Preventing GAGs from
binding to the proteoglycan core protein with para-nitrophenyl xyloside (PNPX) in the
newly forming cartilage matrix of chondrocytes cultured in alginate resulted in less
collagen deposition and a decrease in stiffness and ability to hold water
(Bastiaansen-Jenniskens 2008b). The difference between these two studies is that in
the explant study collagen was already deposited and cross-linked, whereas in the
alginate cultures no collagen was yet deposited when GAG incorporation was
inhibited. Both studies however indicate that modulating GAGs can influence the
collagen network in an already existing cartilage matrix or during new cartilage matrix
synthesis.
Most of the approaches mentioned above focused on modulating one matrix
component (either collagen or proteoglycans). However, increasing collagen
synthesis alone is not sufficient for cartilage repair. Because of the differential and
combining IGF1, BAPN and PNPX in one experiment has the potential to stimulate
matrix production and integrative repair more than each single component.
Chondrocytes cultured in alginate were used to examine the effect of the soluble
factors on new matrix production, either alone or in all possible combinations.
Cartilage explants were used to examine the effect of the factors mentioned above
on integration of two existing cartilage matrices.
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2.2 Preparation of cartilage explants for integration study
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2. Materials and Methods
2.1 Cell culture
Articular cartilage was harvested from the metacarpophalangeal joints of calves aged
6–12 months. Chondrocytes were isolated, suspended in alginate in a concentration
of 4 x 106 cells per mL of alginate, and alginate beads were made as described
previously (Jenniskens 2006). Beads were cultured in a six-well plate (BD Falcon,
Bedford, MA, USA), with 75 µl/bead DMEM/F12 supplemented with 10% fetal bovine
serum (GibcoBRL), 50 µg/ml L-asorbic acid 2-phosphate (Sigma), 50 µg/ml
gentamicin and 1.5 µg/ml fungizone (both GibcoBRL). Chondrocytes were cultured
for 21 days in the presence of 25 ng/ml IGF-1, 0.25 mM BAPN and/or 0.25 mM
PNPX based on previous results (Jenniskens 2006; Bastiaansen-Jenniskens 2008a;
Bastiaansen-Jenniskens 2008b) followed by 21 days in control medium. As known
from these previous studies, BAPN inhibits collagen cross-linking and PNPX prevents
incorporation of GAGs into the matrix. In the 21 days of additional culture without
supplements, cross-links had the ability to form and GAGs to incorporate into the
matrix. Culture medium was changed three times a week. Alginate beads were
harvested after 42 days of culture.

Articular cartilage samples were harvested from the metacarpophalangeal joints of
calves aged 6–12 months. Full-thickness cartilage explants of 8 mm diameter and
with a thickness of 0.9–1.2 mm were prepared using a dermal biopsy punch and
scalpel. From the centre of the explants, 3-mm cores were punched out. All samples
(both outer ring and inner core) were incubated for 24 hours in 10 U/ml highly purified
collagenase VII (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) in DMEM-
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YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 6/20
F12 with 10% fetal calf serum based on previous results (Bos 2002; van de Breevaart
Bravenboer 2004; Janssen 2006). From the study of Bos et al (Bos 2002), it was
concluded that there is some loss of matrix proteins by digesting for 24 hours with
highly purified collagenase, but only on the locations where proteoglycans are lost,
i.e. the area of the wound edge. Pre-treatment with collagenase is beneficial for
integration because it increases the number of viable cells at the wound edge
thereby enabling integration of the wound edges. All the cartilage explants were pre-
treated with the collagenase irrespective of the condition. After incubation, the
samples were washed three times for 10 min in culture medium, and the 3-mm inner
cores were reimplanted in their accompanying 8-mm outer rings. Constructs were
cultured in parallel to the alginate beads for 21 days in the presence of 25 ng/ml IGF-
1, 0.25 mM BAPN and/or 0.25 mM PNPX followed by 21 days in control medium.
Constructs were cultured in 1.5 ml DMEM/F12 per construct supplemented with 10%
fetal bovine serum (GibcoBRL), 50 µg/ml L-asorbic acid 2-phosphate (Sigma), 50
µg/ml gentamicin and 1.5 µg/ml fungizone (both GibcoBRL) in the presence of 25
ng/ml IGF-1, 0.25 mM BAPN and/or 0.25 mM PNPX during the first 21 days. After 42
days, constructs were harvested an immediately fixed in 4% phosphate buffered
formalin.

2.3 Biochemical analysis of alginate beads
Alginate beads were digested overnight at 56°C in papain buffer (250 µg/ml papain in
50 mM EDTA and 5 mM L-cysteine). Glycosaminoglycan (GAG) amount in the digest
was quantified using dimethylmethylene blue (DMB) assay (Farndale 1986). The
metachromatic reaction of GAG with DMB was monitored with a spectrophotometer,
and the ratio A530:A590 was used to determine the amount of GAG present, using
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histological evaluation, serial sections were stained with Hematoxylin & Eosin (H&E).
YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 7/20
chondroitin sulfate C (Sigma) as a standard. The amount of DNA in each papain-
digested sample was determined using ethidium bromide with calf thymus DNA
(Sigma) as a standard. High-performance liquid chromatography (HPLC) of amino
acids (hydroxyproline, Hyp) and collagen cross-links (hydroxylysylpyridinoline, HP)
was performed as described previously (Bank 1996; Beekman 1997). The quantities
of cross-links were expressed as the number of residues per collagen molecule,
assuming 300 Hyp residues per collagen triple helix. Three samples were taken of 7
alginate beads per experimental condition for biochemical analyses.

2.4 Histochemical analysis of cartilage explants
Formalin fixed cartilage constructs were embedded in paraffin. To prevent any
negative influence of processing the constructs for histological analysis, separate
cartilage constructs were placed in little porous polymer bags directly after harvesting
prior to fixation in formalin and processing. However, a certain risk of damage during
histological procedure is unavoidable. Therefore we embedded all the samples at the
same time and did the sectioning in a random order to exclude bias. Sections (6 µm)
were cut using a standard microtome. Prior to the histological stainings, sections
were deparaffinated in xylene and rehydrated through graded ethanol. For
To evaluate integration, paraffin sections were stained with a thionine staining. For
each sample we assessed the percentage of total interface length that had a matrix–
matrix connection. A clear distinction could be made between parts with a matrix
connection and parts of the cartilage touching each other but without a clearly
connected matrix, which were scored as parts with a gap. Integration was determined
at both integration sites within a paraffin section. Interface integration percentages
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were obtained by two blinded observers from measurements of three to four different
sections from each sample, resulting in an average value for each interface.

2.5 Statistical analysis
For the alginate bead cultures and the integration study, four pooled cartilage donors
were used. Statistical analysis was performed using GraphPad Prism 5.01
(GraphPad Software, San Diego, CA, USA) software. All data are presented as mean
± standard deviation. Control groups and groups supplemented with PNPX, BAPN
and/or IGF1 were compared with a ANOVA test followed by a post hoc Bonferroni
test.


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The presence of BAPN alone, BAPN with IGF1, BAPN with PNPX and all three
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3. Results
3.1 Chondrocytes cultured in alginate
After 42 days, DNA content, GAG deposition, collagen deposition and collagen cross-
linking were determined in the alginate beads. In the control condition, DNA content
was 0.49 ± 0.03 µg DNA per alginate bead, which was unaffected by the addition of
BAPN, IGF1 and or PNPX during the first 21 days of culture (Figure 1).
4.38 ± 0.39 µg GAG per bead was deposited in the control condition after 42 days of
culture. As expected, this was significantly reduced in the condition with 0.25 mM
PNPX during the first 21 days. Compared to the control condition, GAG deposition
was also less when a combination of PNPX and IGF1 was present, when PNPX and
BAPN were present together, and when all three components were present in the
culture medium. BAPN or IGF1 alone did not influence GAG deposition, rather their
combination resulted in more GAG deposition (Figure 2).
Regarding the collagen deposition, 6.16 ± 0.91 µg was deposited in the control
condition after 42 days. The presence of PNPX during the first 21 days resulted in
less collagen deposition. Inhibition of collagen cross-link formation with BAPN on the
other hand resulted in more collagen deposition, as expected. BAPN together with
IGF1 increased the collagen deposition even more (Figure 3).
factors during the first 21 days resulted in less cross-link formation, in line with the
LOX inhibition by BAPN. Addition of IGF1 alone, PNPX alone or PNPX with IGF1 did
not change the number of collagen cross-links from the control condition, which was
0.69 ± 0.03 HP per collagen triple helix (Figure 4).

3.2 Cartilage integration
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YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 10/20
In parallel to the alginate cultures, cartilage explants were cultured to investigate the
integrative capacity in the presence of PNPX, BAPN and/or IGF1. As in the alginate
cultures, soluble factors were only present during the first 21 days of culture followed
by culture in control medium for an additional 21 days. Typical examples of
integration after 42 days visualised with a thionine staining are shown in figure 5. The
average integration percentage in the control condition was less than 10%. Only
when PNPX, BAPN and IGF1 were combined during the first 21 days, integration of
the two opposing cartilage explants improved to 41.3 ± 31.4% (p < 0.05). Although
not reaching statistical significance, the presence BAPN and IGF1 alone or together
also seems beneficial for integration.
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alginate bead. In addition, excretion of both matrix proteins into the culture medium
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4. Discussion
Many factors have been demonstrated to be able to influence proteoglycan production,
collagen production or both. However, it is not fully known what the optimal balance of
collagen and proteoglycan production is and how they influence each other to form a
functional network.
Our goal was to examine the effect of modulating collagen deposition, collagen
cross-linking and GAG deposition simultaneously during cartilage matrix production
and cartilage integrative repair, thereby creating a wider insight in the role of
proteoglycans and collagen in cartilage repair process.
In the alginate cultures, inhibition of collagen cross-linking with BAPN in combination
with promotion of matrix synthesis using IGF1 was most beneficial for matrix
deposition. Additional PNPX was always detrimental for matrix deposition. For
integration, the combination of BAPN, IGF1 and temporary prevention of
proteoglycan formation with PNPX was most beneficial.
Inhibition of GAGs from binding to the proteoglycan core protein not only resulted in
less GAG deposition in the alginate bead, but also in less collagen deposition. This is
in concordance to our previous study were the presence of PNPX during a culture
period of 35 days resulted in less proteoglycan and collagen deposition in the
was higher due to the absence of an intact matrix network (Bastiaansen-Jenniskens
2008b). Because of the absence of this network and the loss of interaction with other
matrix components, combining PNPX with BAPN and/or IGF1 could not counteract
the effects seen with PNPX alone. Earlier we found that the inhibition of collagen
cross-linking in an alginate culture for 21 days and longer results in higher collagen
deposition then when cross-links are formed. During this culture period, proteoglycan
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1983) and therefore extra time is also needed for LOX to be produced again. Based
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deposition was unaffected (Bastiaansen-Jenniskens 2008a). Even though cross-link
inhibition by BAPN stimulates collagen synthesis, the retention of collagen is very low
because of the absence of a proteoglycan network. Previously, we and others
showed stimulating effects of IGF1 on collagen and proteoglycan deposition (Blunk
2002; Mauck 2003; De Mattei 2004; Jenniskens 2006). In the present setup, culture
with IGF1 alone for 21 days did not significantly stimulate matrix deposition after 42
days, whereas IGF1 did previously already after 21 days (Jenniskens 2006).
However, the combination IGF1 and BAPN increased GAG deposition which is
probably attributable to the presence of IGF1 since BAPN does not affect the GAG
deposition. Even though IGF1 might have also stimulated GAG and collagen
deposition when combined with PNPX or with PNPX and BAPN, GAGs were
prevented from incorporation into the matrix. A possible positive effect of IGF1 on
collagen synthesis was counteracted because of low collagen retention in the
alginate bead. In every condition where the cross-link inhibitor BAPN was present for
the first 21 days, less collagen cross-links were present even though the culture
continued for 21 days after removal of BAPN. This might be explained by the fact that
after removal of BAPN, it takes three weeks for mature HP cross-links to be formed
(Ahsan 2005). In addition, BAPN binds irreversibly to lysyl oxidase (LOX) (Tang
on our previous experiments, we expect that the number of collagen cross-links will
be equal in all conditions when a longer culture period is used (Bastiaansen-
Jenniskens 2008a).
The combination of BAPN and IGF1 seems most beneficial for matrix synthesis when
no matrix is formed yet, as in the alginate cultures. When aiming at the integration of
two existing cartilage matrices, cross-link inhibition with BAPN and stimulation of
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but without IGF1 was also not sufficient because of the absence of increased
YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 13/20
matrix production with IGF1 is not sufficient to significantly improve integration.
Transient inhibition of GAG incorporation in the matrix with PNPX in combination with
BAPN and IGF1 did improve the integration. In these experiments, stimulation of
matrix production alone with IGF1 had no effect. Transient inhibition of collagen
cross-link formation could have resulted in more collagen synthesis, and in addition in
better penetration of the newly formed collagen fibres in the opposing tissue that was
partly depleted of GAGs following injury (Bos 2001). The latter is more likely since
previous experiments with BAPN and cartilage explants did not result in more
collagen synthesis (McGowan 2005). The study by McGowan et al is also in
concordance with the theory of better integration after BAPN treatment. The addition
of PNPX and the resulting absence of GAG attachment to the proteoglycan core
protein might have similar effects as seen with inhibition of cross-link formation.
Without an intact proteoglycan network at the edges of the cartilage explants, newly
formed collagen fibres might penetrate better into the opposing cartilage tissue.
Because of the absence of a proteoglycan network, the collagen network might also
have a better integrity (Asanbaeva 2007). This explains why the presence of IGF1
with PNPX or BAPN did not improve integration since the newly formed collagen
stimulated by IGF1 could not penetrate into the cartilage. PNPX together with BAPN
collagen production.
When cartilage transplantations are performed, the surgeon removes the damaged
cartilage first. Removing the damaged cartilage will create a fresh wound area,
similar to our experimental condition. We hypothesize that the integrative capacity of
such an area is different from a long lasting wound area and we are therefore
convinced that our explant model is close to the application in this respect.
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YM Bastiaansen-Jenniskens; combining IGF-1, BAPN, PNPX 14/20
The cartilage explants used for integration were cultured for 6 weeks in vitro. In an
earlier study, we investigated the effect of improved integration on mechanical
properties of the cartilage-cartilage interface (van de Breevaart Bravenboer 2004).
There we observed a relation between the area of integration on histology and the
mechanical properties of the interface. Since the previous study is performed after an
in vivo culture period and our current study evaluates after in vitro culture, it is difficult
to extrapolate these results. Earlier attempts in the lab have demonstrated us that
after in vitro culture the bonding is not strong enough to measure reliably in our
system. However, the improved histological integration in the present studies looks
promising and suggests that when a construct is placed in vivo after modulation with
our soluble factors (IGF1, PNPX and BAPN), integration might lead to functional
cartilage.
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