Aggrecan catabolism during mesenchymal stromal cell in vitro chondrogenesis

Marıa Lucıa Gutierreza*, Johana Marıa Guevaraa, Olga Yaneth Echeverria, Diego Garzon-Alvaradob and

Luis Alejandro Barreraa

aInstitute for the Study of Inborn Errors of Metabolism, Pontificia Universidad Javeriana, K 7 No. 43�82 Lab 305A Edificio JesusEmilio Ramirez, Bogota, Colombia; bBiomimetics Laboratory, Biotechnology Institute, Universidad Nacional de Colombia, K 30No. 45�03 Edificio 407 Oficina 203, Bogota, Colombia

(Received 5 April 2013; accepted 4 June 2013)

During skeleton formation, mesenchymal cells condense and differentiate into chondrocytes in a process known aschondrogenesis. Glycosaminoglycans (GAGs), main components of aggrecan in the extracellular matrix (ECM),have an important role in this process. An in vitro simplified system has been devised to study chondrogenesis usingmesenchymal progenitor cells. Although the capacity of mesenchymal stromal cells to differentiate into thechondrogenic lineage is well established, there is a lack of knowledge with respect to lysosomal enzyme activityduring the chondrogenic process. To further understand GAG’s catabolic activities during in vitro chondrogenesis, weevaluated three lysosomal enzymes. Chondrogenic differentiation was demonstrated by Alcian blue positive stainquantified by a grading system using ImageJ. Enzyme activity for N-acetylgalactosamine-6-sulfate-sulfatase duringchondrogenic induction decreased significantly with time of culture; b-galactosidase enzyme activity had a similartendency of temporal activity. On the contrary, b-glucuronidase enzyme activity decreased from the first to secondweek of induction, but remained the same during the third week of culture. Aggrecan’s immunohistochemistry valuesfor aggregates under chondrogenic induction revealed a similar temporal pattern to that of N-acetylgalactosamine-6-sulfate-sulfatase and b-galactosidase enzyme activity. This work has contributed to the evaluation of enzymeactivities associated with GAG degradation, critical component of cartilage ECM. These findings are relevant inunderstanding the role of enzymes responsible for degradation of molecules predominantly synthesized inthe chondrogenic differentiation process. A better understanding of the roles of these enzymes during developmentcould help elucidate further association of deficiencies of these enzymes in skeletal pathologies, primarilychondrodysplasias.

Keywords: glycosaminoglycans; mesenchymal stromal cells; chondrogenic differentiation; lysosomal enzymes

1. Introduction

During embryological skeleton development chondro-

cytes derive from mesenchymal cells that migrate to

specific sites and differentiate into chondrocytes in a

process known as chondrogenesis. Chondrocytes in-

habit a hyperosmotic and acidic environment resulting

from a polyanionic extracellular matrix (ECM), prin-

cipally composed of collagen type II and glycosami-

noglycans (GAGs) (Goldring et al. 2006; Han et al.

2011). Chondrocytes synthesize their surroundings,

and their effectiveness as homeostatic regulators par-

tially depends on their capability to synthesize and

catabolize ECM components. Any alteration in this

balance is associated with diseased cartilage. Although

cartilage GAG synthesis has been described (DeLise et

al. 2000; Zhang et al. 2003; Goldring et al. 2006;

Gotting et al. 2007; Cameron et al. 2009; Ghone &

Grayson 2012), little is known about GAG catabolism

during chondrogenesis (Settembre et al. 2008; Ratzka

et al. 2010). The purpose of this study was to evaluate

the pattern of specific lysosome enzyme activities,

during in vitro chondrogenesis, to shed light on their

biological significance.

Embryonic appendicular skeleton formation is

achieved through chondrogenesis. These mechanisms

are delicately controlled in space and time by cellular

interactions, growth and differentiation factors, and

other environmental elements (Sandell & Adler 1999;

Sekiya et al. 2002; Kronenberg 2003). During skeleton

formation, mesenchymal cells condense and differenti-

ate into chondrocytes forming a cartilage mold of

future bones. Bone results as the replacement of the

cartilaginous intermediate by endochondral ossifica-

tion (Kronenberg 2003). Throughout chondrogenesis,

the ECM plays a pivotal role in morphogenesis and

development. Growth factor availability is controlled

by the ECM resulting in regulation of cell differentia-

tion, proliferation, adhesion, and migration (DeLise

et al. 2000; Lin et al. 2006). The chondrogenic mold is

characterized by the synthesis of collagen type II and

aggrecan, proteoglycans (PGs) composed of a core

protein and the GAGs keratan sulfate (KS) and

chondroitin sulfate (CS) linked in a perpendicular

manner (Gotting et al. 2007; Lamoureux et al. 2007).

However, within the cartilage matrix other sulfated

GAGs such as heparan sulfate (HS), dermatan sulfate

(DS), and nonsulfated hyaluronan also function as

*Corresponding author. Email: mlgutierrez@javeriana.edu.co

DEVELOPMENTAL

BIO

LOGY

Animal Cells and Systems, 2013

Vol. 17, No. 4, 243�249, http://dx.doi.org/10.1080/19768354.2013.812537

# 2013 Korean Society for Integrative Biology

stabilizers, cofactors, and/or corepressors for growth

factors, cytokines, and chemokines (DeLise et al. 2000).

Approaches to understand chondrogenesis have

used mice and chick organ cultures of embryonic limbs

(Sekiya et al. 2002; Goldring et al. 2006). In the mouse

embryo, global transcriptome analysis has identified

cartilage formation to occur at embryonic days 11.5

(precondensation of mesenchymal cells), 12.5 (conden-sation), and 13.5 (differentiation) (Cameron et al.

2009). Due to the complexity of embryonic chondro-

genesis, for over a decade, a simplified in vitro approach

has been used to study mesenchymal stromal cell

(MSC) chondrogenic differentiation. In vitro aggregate

culture technique allows direct study of the chondro-

genic differentiation process. It duplicates cellular

condensation and induces differentiation by culturingcells in conditioned media (Yoo et al. 1998). MSCs are

multipotent progenitors of connective tissue, with the

potential to differentiate in vitro and in vivo into

mesodermal lineages: adipo-, osteo-, and chondrogenic

(Baksh et al. 2004). They have been isolated from many

tissues,and subcutaneous adipose tissue is an attractive

source with known chondrogenic differentiation poten-

tial (Erickson et al. 2002; Zuk et al. 2002).Although the capacity of MSC to differentiate into the

chondrogenic lineage is well established, there is a lack

of knowledge with respect to GAG catabolism during

chondrogenesis. To further understand the enzyme activ-

ity responsible for GAG degradation, we evaluated three

lysosomal enzymes using aggregate culture technique with

human adipose-derived MSCs undergoing chondrogenic

differentiation. We characterized the enzymatic activity ofN-acetylgalactosamine-6-sulfate-sulfatase (GALNS), b-

galactosidase, and b-glucuronidase during three-week

aggregate culture. GALNS catalyzes the hydrolysis of

sulfate ester bonds in chondroitin-6-sulfate (C6S), and

galactose-6-sulfate in KS (Bielicki et al. 1995). b-

Galactosidase carries out the subsequent degradation of

KS (Ohto et al. 2012), and hydrolysis of the terminal

nonreducing b-D-glucuronide in HS, DS, and CS iscarried out by b-glucuronidase (Neufeld & Muenzer

2001; Rowan et al. 2012). The purpose of this study was

to evaluate GAG catabolism during in vitro chondro-

genesis to understand possible temporal changes in

enzyme activity during MSC in vitro chondrogenesis of

specific lysosome enzymes, and to better define the

importance of GAG catabolism during chondrogenesis.

2. Methods

We hypothesized that enzyme activity should have a

temporal pattern of activity during in vitro chondro-

genesis, likely to be associated to the substrate catabo-

lised. To verify this hypothesis, first we isolated MSCsform adipose tissue and characterized them according

to the minimal criteria established by the ISCT

(Dominici et al. 2006).

2.1. MSC isolation and characterization

Samples were obtained after informed signed consent

from three females (aged from 24 to 38) undergoingcosmetic surgery with the approval of the Bioethics

Committee at the Pontificia Universidad Javeriana.

Adipose tissue MSCs were isolated as previously

described with slight modifications (Zuk et al. 2001).

Briefly, samples were washed and digested with collage-

nase type I. A stromal vascular fraction (SVF) was

obtained and resuspended in a-minimum essential

media (MEM) without ribonucleosides or ribonucleo-tides (Sigma�Aldrich, St. Louis, MO, USA) with 10%

FBS, antibiotics, and antimycotic (complete media), and

seeded in 100-mm tissue culture. Tissue culture plates

were incubated at 378C with 5% CO2 and humidified

atmosphere. Media were changed 24 h after plating.

MSCs from donors were cultured in complete media

below passage seven. Cells were immunophenotyped

and characterized as previously described (Gutierrez etal. 2012). For aggregate culture of 250�103 cells

passage, two were placed in a polypropylene tube and

centrifuged to form a pellet. Twenty-four hours after

centrifugation induction was initiated. The cells were

cultured in complete media or chondrogenic differen-

tiation media (Lonza, Walkerville, MD) supplemented

with BMP-6 (Sigma) and TGF-b3 (Invitrogen) at 10 ng/

ml, respectively. Media was changed twice a week for21 days. Chondrogenesis was confirmed by Alcian blue

positive stain percentage evaluated by ImageJ analysis,

as previously described (Gutierrez et al. 2012).

To determine possible temporal changes in enzyme

activity during MSC aggregate culture, cells were

maintained for three weeks in either complete media

or chondrogenic differentiation media to determine

their pattern of enzyme activity.

2.2. Biochemical analyses

For intracellular GALNS, b-galactosidase and b-glucur-

onidase enzyme activity aggregates were sonicated in

300-ml (155 mM) saline solution for 20 seconds in three

cycles on ice. GALNS enzyme activity was measuredusing the fluorogenic substrate 4-methylumbelliferyl-

b-galactopyranoside-6-sulfate (TRC, Canada), as des-

cribed by van Diggelen with modifications (van Diggelen

et al. 1990). For b-galactosidase and b-glucuronidase

enzyme activity, the fluorogenic substrates 4-methylum-

belliferyl-b-D-galactoside and 4-methylumbelliferyl-b-D-

glucuronide (both form Sigma) were used, respectively.

Activity was determined according to Shapira et al. (1989).GALNS specific activity in aggregate culture was calcu-

244 M.L. Gutierrez et al.

lated by normalizing volumetric activity (nmol/h/ml) to

GALNS quantity determined by indirect enzyme-linked

immunosorbent assay (ELISA) (ng/ml). Indirect ELISA

was performed from aggregate lysates as reported by

Rodriguez et al. (2010). GALNS antibody was kindly

provided by Dr. Shunji Tomatsu (Skeletal Dysplasia Lab

Pediatric Orthopedic Surgery, Nemours/Alfred I. duPont

Hospital for Children, Wilmington, DE, USA).To verify aggrecan’s synthesis during aggregate

culture, IHC was carried out during the three-week

culture.

2.3. Aggrecan IHC values

As previously described, aggrecan’s IHC values were

obtained using a semiautomatic grading system to

evaluate in vitro chondrogenesis (Gutierrez et al. 2012).

2.4. Statistical analysis

Results are presented as mean9standard error (n�3

with replicates per sample), and analyzed using Stu-

dent’s t-test to compare MSCs cultured in complete

media vs. cells cultured in chondrogenic differentiationmedia. Analysis of variance (ANOVA) was used to

compare differences among groups. Differences were

considered significant at pB0.05. The SPSS for

Windows (IBM version 18) and Statistix (Analytical

software version 9.0) were used for statistical analysis.

3. Results

3.1. MSC characterization

Mesenchymal stromal cells isolated from processed

lipoaspirate (PLA) complied with all criteria to identify

them as MSCs according to the International Society

for Cellular Therapy (ISCT) (Dominici et al. 2006), aspreviously described (Gutierrez et al. 2012).

3.2. Aggregate culture phenotype

To determine chondrogenic differentiation, Alcian blue

positive stain percentage carried out by ImageJ analysis

revealed a significantly higher percentage in aggregate

cultures in chondrogenic differentiation media com-

pared to complete media. In addition, we observed a

decreasing trend over time for Alcian blue stain in

aggregates cultured in chondrogenic differentiation

media (Table 1).

3.3. Aggrecan immunohistochemistry (IHC) values

We observed a decreasing tendency with time foraggrecan’s marker assessment percentage for aggregates

cultured in chondrogenic differentiation media. On the

contrary, aggregates cultured in complete media had an

increased percentage with increasing time of culture

(Table 2), as previously determined by ImageJ analysis

(Gutierrez et al. 2012).

3.4. Lysosome enzyme activity

GALNS volumetric enzyme activity (nmol/h/ml) sig-

nificantly decreased in a temporal manner during

chondrogenic induction, with highest activity at early

events of chondrogenic differentiation. GALNS en-

zyme activity was significantly different for spheres

cultured in complete media vs. spheres cultured in

chondrogenic differentiation media (Figure 1).

GALNS specific activity was determined by normal-

izing GALNS volumetric activity (nmol/h/ml) to

GALNS determined by indirect ELISA (ng/ml). Specific

activity demonstrated a significant decrease of GALNS

Table 1. Alcian blue positive stain percentage obtained by

using ImageJ analysis.

Complete

media

Significance

levels for

CM vs.

CDM

Chondrogenic

differentiation

media

Significance

levels for

CDM

between

weeks

Week 1 2.7690.67 *** 19.5091.15 ***

Week 2 1.3690.33 *** 16.2591.09 ***

Week 3 3.7690.40 ** 8.7791.13 ***

Cells cultured in aggregates in complete media or chondrogenicdifferentiation media during the first, second and third week ofculture. Percentage was determined as previously described(Gutierrez et al. 2012).One way ANOVA **pB0.01 (CMW3 vs. CDMW3).***pB0.001 (CMW1 vs. CDM WK1, CMW2 vs. CDM W2.CDMW1 vs. CDMW3. CDMW2 vs. CDMW3).CM, complete media; CDM, chondrogenic differentiation media;WK, week.

Table 2. Aggrecan’s marker assessment determined by ImageJ

analysis.

Complete

media

Significance

levels for CM

between weeks

Chondrogenic

differentiation

media

Week 1 0.2090.05 * 0.6990.55

Week 2 0.3490.12 0.5290.16

Week 3 1.1990.28 * 0.2790.06

Cells cultured in aggregates in complete media or chondrogenicdifferentiation media for 7 (Week 1), 14 (Week 2), or 21 days (Week 3).Aggrecan marker assessment percentage was significantly higher foraggregates in complete media during the third week of culturecompared to the first week (One way ANOVA *pB0.05 WK1 vs.WK3).WK, week.

Animal Cells and Systems 245

(nmol/h/ng) with time for aggregates undergoing chon-

drogenic differentiation (Figure 2).

Analysis of b-galactosidase volumetric enzyme

activity in aggregates cultured in chondrogenic

differentiation media was significantly higher com-

pared to spheres cultured in complete media for the

first and second week of culture. For spheres cultured

in chondrogenic media, enzyme activity had a pattern

with a decreasing tendency, resembling that of GALNS,

during the three weeks of culture (Figure 3A).

b-Glucuronidase activity, a lysosomal enzyme

which catabolizes DS, HS, and CS, had a more uniform

enzyme activity for cells under chondrogenic induction

during the three weeks of culture compared to enzymes

responsible for initiation of KS and C6S catabolism.

For aggregates cultured in complete media, enzyme

Figure 3. (A) Beta galactosidase volumetric enzyme activity.

Activity was calculated in aggregates cultured for three weeks

in complete (white bars) and chondrogenic media (black

bars). Enzyme activity for spheres in complete media was

significantly lower compared to spheres in chondrogenic

differentiation media (Student’s t-test *pB0.05, ***pB

0.001) for the first and second week of culture. (B) Beta

glucuronidase volumetric enzyme activity. Activity was

calculated in aggregates cultured for three weeks in complete

and chondrogenic differentiation media. Enzyme activity for

spheres in complete media (white bars) was significantly

lower compared to spheres in chondrogenic differentiation

media (black bars) for the second and third week of culture

(Student’s t-test *** pB0.001).

Figure 1. GALNS enzyme activity. Volumetric GALNS

activity (nmol/h/ml) was calculated in aggregates cultured

for one, two, or three weeks in complete (white bars) or

chondrogenic differentiation media (black bars). Enzyme

activity was significantly different for spheres in complete

media vs. chondrogenic differentiation media (Student’s t-test

**pB0.005). Enzyme activity from spheres in chondrogenic

differentiation media cultured for one week was significantly

higher compared to spheres cultured for two or three weeks.

In addition, aggregates under chondrogenic induction during

the second week of culture had a higher activity compared to

aggregates during the third week of culture (ANOVA *pB0.05,

***pB0.001).

Figure 2. GALNS specific activity (nmol/h/ng). Enzyme

activity was calculated from aggregates cultured for 7, 14,

or 21 days in complete media (white bars) or chondrogenic

differentiation media (black bars). Specific activity was

significantly higher for aggregates cultured in chondrogenic

differentiation media during the first week of culture com-

pared to the third week (ANOVA *pB0.05). Significant

differences were also observed between aggregates in com-

plete compared to those cultured in chondrogenic differentia-

tion media during the first and second week of culture

(Student’s t-test *pB0.05).

246 M.L. Gutierrez et al.

activity was comparable to b-galactosidase during the

three weeks of culture. Complete media aggregates

had a significantly lower enzyme activity compared

to aggregates in chondrogenic differentiation media

during the second and third week of culture (Figure

3B). In summary, b-glucuronidase enzyme activity

during chondrogenic induction did not seem to have

a temporal decrease during the three weeks of culture.Collectively, our results in general demonstrate

greater enzyme activities in aggregates cultured in

chondrogenic media compared to complete media. In

chondrogenic differentiation media, the pattern of

catabolic activity for GALNS and b-galactosidase,

enzymes involved in KS and C6S catabolism during

the three weeks of culture, was a gradual decrease from

the first to third week of culture. It is noteworthy thatGALNS specific enzyme activity was significantly

higher during the first week of culture in chondrogenic

differentiation media compared to the third week of

aggregate culture. On the contrary, enzyme activity for

b-glucuronidase was more uniform during the three-

week culture. In addition, based on the scores obtained

from a semiquantitative IHC grading analysis using

ImageJ, aggrecan’s marker assessment percentage wasgreater during the early events of chondrogenic differ-

entiation with a gradual decrease with time.

4. Discussion

Cartilage ensued from mesenchymal differentiationcontains large amounts of GAGs in the ECM, and

their proper catabolism provides tissue homeostasis. A

broad spectrum of skeletal abnormalities has been

identified as a result of deficient enzymes unable to

catabolise GAGs adequately, therefore underlying their

biological importance (Settembre et al. 2008). The aim

of this study was to define possible temporal enzyme

activity patterns for three lysosomal enzymes: GALNS,b-galactosidase, and b-glucuronidase in a well-estab-

lished model for in vitro chondrogenesis (Yoo et al.

1998).

Chondrogenesis was promoted by known chondro-

genic inducers (Bohme et al. 1992; Estes et al. 2006;

Rich et al. 2008), and differentiation was confirmed by

Alcian blue positive stain percentage. Spheroids cultured

for three weeks in chondrogenic differentiation mediahad a significant higher percentage compared to aggre-

gates in complete media. Thus, confirming that inducers

had an effect on total GAG synthesis during aggregate

culture suggests cells in chondrogenic differentiation

media were undergoing differentiation.

We set out to assay activities for three lysosomal

enzymes responsible for GAG degradation during in

vitro chondrogenesis: GALNS, b-galactosidase, andb-glucuronidase. Deficiencies in these enzymes result

in skeletal abnormalities with shortened long bones

through mechanisms largely unknown in patients with

Morquio A (Mucopolysaccharidosis IVA), Morquio B

(Mucopolysaccharidosis IVB), and Sly disease

(Mucopolysaccharidosis VII), respectively. These dis-

orders are called mucopolysaccharidoses, since GAGs

were previously named as mucopolysaccharides. Defi-

ciency of the previously mentioned lysosomal enzymes

interrupts GAG degradation and results in intralyso-

somal GAG accumulation (Neufeld & Muenzer 2001).Changes in lysosomal enzyme activities responsible

for GAG degradation related to the process of

chondrogenesis either in vivo or in vitro have not been

reported. A study by Ratzka et al. described expression

patterns of sulfatase genes in the developing mouse

embryo. From their work, a total of six sulfatases

were found in the developing skeleton: arylsulfatase

B (ArsB), ArsI, ArsJ, glucosamine-6-sulfatase, Sulf1,

and Sulf2. Nonetheless, Galns, b-galactosidase, or

b-glucuronidase was not described by them. For Galns

it was reported that it was ubiquitously expressed in

tissues on days 12.5 and 14.5 of embryological devel-

opment, not on day 13.5, when differentiation takes

place (Ratzka et al. 2010). Furthermore, a global

comparative transcriptome analysis of cartilage forma-

tion, in vivo, did not describe the role of enzymes

associated with aggrecan’s catabolism, a predominant

cartilage matrix component (Cameron et al. 2009). This

association is relevant, since a defect in any one of

these three enzymes is manifested as a skeletal disease

(Neufeld & Muenzer 2001). For example, chondrocytesfrom Morquio A patients have phenotypic changes as a

consequence of undegraded KS manifested in terms

of altered gene expression resulting in a chondrodys-

plasia (Dvorak-Ewell et al. 2010). Furthermore, De

Franceschi and colleagues described decreased aggre-

can mRNA in samples obtained from femoral condyle

of the knee in two severe Morquio A patients (De

Franceschi et al. 2007).

Aggrecan is composed of a small KS domain and a

larger CS domain (Rodriguez et al. 2006), and makespart of the avascular ECM synthesized during chon-

drocyte differentiation from mesenchymal stem cells

(Blair et al. 2002). GALNS catalyzes the first step of

intralysosomal KS or C6S catabolism (Pshezhetsky &

Potier 1996; Tomatsu et al. 2011). The following step in

KS’ degradation is carried out by b-galactosidase

(Neufeld & Muenzer 2001; Ohto et al. 2012). Human

adipose MSC chondrogenic differentiation studies have

revealed that aggrecan’s expression is restricted to early

events, as early as the first week of induction, with a

decrease after the second week of differentiation(Erickson et al. 2002; Zuk et al. 2002; Estes et al.

2006). Most chondrogenesis studies have focused on

aggrecan’s synthesis, which is the characteristic of

Animal Cells and Systems 247

mesenchymal cell differentiation into chondrocytes and

proliferation. On chondrocytes that have fully differ-

entiated, its synthesis stops (Goldring et al. 2006). It is

possible that GALNS and b-galactosidase activities are

highest when aggrecan synthesis initiates, and decline

concomitantly as aggrecan is not synthesized again.

From our results, we observed during the course of

three-week culture a gradual decrease in GALNS and b-

galactosidase enzyme activity. This similar pattern of

enzymatic activity decline might be explained by GALNS

and b-galactosidase close relationship, as they form part

of the multienzyme complex of lysosomal hydrolases and

are related to KS catabolism, main component of

aggrecan (Pshezhetsky & Potier 1996). On the other

hand, b-glucuronidase related to DS, HS, and CS

catabolism, displayed a more homogenous pattern of

enzyme activity during the three weeks of culture in

chondrogenic media (Metcalf et al. 2009).

Last, aggrecan’s IHC results revealed a gradual

decrease with time for cells undergoing chondrogenic

differentiation. The pattern of aggrecan’s marker

assessment percentage as determined by semiquan-

titative analysis is parallel to that of GALNS and

b-galactosidase enzyme activities during the three

weeks of chondrogenic induction. We suggest that

differences in the patterns of enzyme activities observed

during in vitro chondrogenesis could be associated to

the substrates degraded. Further studies to confirm this

association are needed.

In conclusion to our knowledge, this is the first

report to determine enzymatic activities associated

with ECM GAG degradation, specifically GALNS,

b-galactosidase, and b-glucuronidase using aggregate

culture technique during chondrogenic differentiation.

GALNS and b-galactosidase enzyme activities decreased

in a temporal manner during the in vitro chondrogenesis

process. These findings are relevant in understanding

the role of enzymes responsible for degradation of

molecules predominantly synthesized during the chon-

drogenic process, in particular, molecules specific to

cartilage tissues such as KS and CS. Further studies

identifying its participation during in vivo chondro-

genesis could shed some light on chondrodysplasia

therapeutic approaches.

Acknowledgments

This work was funded by Colciencias Grant 1203440820438.We would like to thank Dr Felipe Amaya M.D. for providinglipoaspirate samples.

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