J. Biomedical Science and Engineering, 2010, 3, 148-159
doi:10.4236/jbise.2010.32020 Published Online February 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online February 2010 in SciRes. http://www.scirp.org/journal/jbise
Senescence of human vocal fold fibroblasts in primary culture
George M. Adams1, Chet C. Xu1, Roger W. Chan1,2
1Otolaryngology, Head and Neck Surgery, University of Texas Southwestern Medical Center, Dallas, USA;
2Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, USA.
Received 1 October 2009; revised 18 December 2009; accepted 21 December 2009.
ABSTRACT
This study examined the replicative senescence of
primary-culture human vocal fold fibroblasts, in terms
of changes in gross chromosomal structure with sub-
culturing, and population doubling time (PDT). The
mRNA expressions of 14 target genes were also ex-
amined. The objectives were to identify the onset of
senescence for establishing the acceptable limit of
sub-culturing, and to better understand the effect of
cellular aging on matrix protein regulation in the
vocal fold. Gross chromosomal changes in vocal fold
fibroblasts from a 58-year-old woman were karyo-
typed with Giemsa stain. Proliferation of the fibro-
blasts was determined with cell recovery and PDT
calculation. Transcript levels of the target genes were
found by RT-PCR. Onset of significant chromosomal
anomalies was seen with passage 5. For mRNA ex-
pressions, significant increases with passaging were
observed in collagenase, macrophage elastase, lysyl
oxidase and fibromodulin, whereas significant down-
regulation was detected in decorin, procollagen I,
hyaluronic acid-synthase 2 and collagen III. This
modulation pattern suggested that fibroblasts un-
derwent in vitro aging, consistent with the significant
increase in PDT. The inception of senescence did not
occur until passage 5. These findings may facilitate
the development of representative in vitro models for
testing tissue engineering approaches involving pri-
mary-culture fibroblasts.
Keywords: Larynx; Extracellular Matrix; Cell Proliferation;
Replicative Senescence; Tissue Engineering
1. INTRODUCTION
A better understanding of the biology of vocal fold fi-
broblasts is fundamental for establishing representative
and consistent in vitro models of the vocal fold lamina
propria extracellular matrix (ECM), where fibroblasts
are the predominant cell type [1,2]. Such in vitro models
are critical for the development of tissue engineering
approaches for the treatment of a variety of vocal pa-
thologies. Tissue engineering, or regenerative medicine
approaches involve the fabrication of tissue substitutes
that can be used to surgically augment, replace, and
functionally repair tissue defects in the lamina propria,
e.g., vocal fold scarring, atrophy, sulculs vocalis, and
tissue deficiencies left with the surgical removal of tu-
mors [3]. Vocal fold fibroblasts are responsible for the
homeostatic expressions of key ECM components of the
lamina propria, including fibrous proteins (e.g., collagen,
elastin), proteoglycans (e.g., decorin, fibromodulin, ver-
sican, biglycan), glycosaminoglycans (e.g., hyaluronic
acid), and structural glycoproteins (e.g., fibronectin)
[1,2]. Expressions of these ECM proteins contribute to
the optimal tissue viscoelastic properties crucial for func-
tional voice production, since the lamina propria is the
primary tissue layer responsible for flow-induced self-
oscillation of the vocal fold [4]. The structural and func-
tional integrity of the ECM depends on the proliferation,
metabolic activities, and turnover rate of vocal fold fi-
broblasts. ECM changes have often been studied with
cultured vocal fold fibroblasts, yet it is well established
that fibroblasts in vitro are influenced by a phenomenon
known as replicative senescence, an in vitro form of ag-
ing somewhat parallel to the senescent processes in vivo
[5]. Cells undergo replicative senescence after repeated
subculturing for a number of passages. Replicative se-
nescence is characterized by decreased cellular prolif-
eration, resistance to mitosis, and avoidance of pro-
grammed cell death or apoptosis [5,6].
Thibeault et al. examined the karyotypes of normal
vocal fold fibroblasts and tracheal scar fibroblasts from
primary culture, across different passages (passages 3, 4,
5, 6 and 10) [7]. Normal vocal fold fibroblasts displayed
multiple chromosomal abnormalities including loss of
the Y chromosome, translocations, multiple rearrange-
ments, and tetraploidy of all chromosomes excluding the
Y chromosome. With tracheal scar fibroblasts, no aber-
rations were detected in the early passages while 15 of
20 cells analyzed in the late passage showed tetraploidy
in all but the sex chromosome. While these data pro-
vided an initial understanding of chromosomal abnor-
malities in primary vocal fold fibroblasts, the onset of

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149
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these abnormalities with passaging was not known, par-
ticularly in early passages (before passage 3). This study
attempted to determine the inception of these anomalies
using detailed cytogenetic analyses of early and late
passages (passages 1-5, and passage 10) of normal pri-
mary-culture human vocal fold fibroblasts. The expres-
sions of key matrix proteins in the lamina propria ECM
and their protein precursors, as well as those of the ma-
trix-synthesizing and matrix-degrading enzymes at the
mRNA level were examined by reverse transcriptase-
polymerase chain reaction (RT-PCR). The rate of prolif-
eration of the fibroblasts was also investigated by meas-
urement of the population doubling time (PDT). Our
goals were: 1) to identify the onset of senescent mecha-
nisms for establishing an acceptable limit of sub-cultur-
ing; and 2) to better understand the effect of cellular ag-
ing on the homeostatic regulation of matrix proteins in
the vocal fold lamina propria, so that one could establish
representative, consistent in vitro models of the ECM
based on the acceptable limit of sub-culturing. Such
models can serve as a platform for evaluating the impact
of tissue engineering approaches on functional tissue
remodeling and reconstruction, e.g., approaches based
on the use of acellular bioscaffolds, polymeric substrates,
or growth factors [8,9,10].
2. MATERIALS AND METHODS
2.1. Vocal Fold Tissue Culture
Primary-culture vocal fold fibroblasts were obtained
from the vocal ligament (intermediate and deep layers of
the lamina propria) of a larynx excised from a 58-year-
old Hispanic female who underwent total laryngectomy
due to papillary columnar thyroid carcinoma. The la-
ryngeal specimens were examined by an otolaryngolo-
gist as well as by a pathologist, and were certified to be
free of cancerous tissue, i.e., without any epithelial to
mesenchymal transition [11]. The protocols for the pro-
curement of the laryngeal specimens and the subsequent
experimental procedures were approved by the Institu-
tional Review Board of UT Southwestern Medical Cen-
ter. Using primary explant techniques as described in Xu
et al. the specimens were disinfected with 70% ethanol,
washed twice with phosphate-buffered saline (PBS),
dissected using phonomicrosurgical instruments and cut
into 1- to 2-mm3 sections [8]. The tissue sections were
then placed in a 24-well plate (one section per well) and
supplemented with DMEM fortified with 20% fetal bo-
vine serum (FBS) and 2% penicillin/streptomycin. Pri-
mary fibroblasts reproduced and migrated from the tis-
sue sections over a two-week period prior to being sub-
cultured, or passaged. The primary cells were authenti-
cated as conventional vocal fold fibroblasts according to
their morphological characteristics under light micros-
copy [12]. For sub-culturing, cells were grown to 60%
confluence and treated with trypsin-EDTA for 3-4 min at
37°C with 5% CO2. An equal volume of complete media
was then added to the trypsinized suspension prior to
centrifuging for 3 min at 1500 rpm. After removal of the
supernatant, the pellet was resuspended in 8 ml of com-
plete media with 1 ml added to a 75-cm2 tissue culture
flask (T-75), at 1:8 dilution. DMEM was then added
(20-25 ml) to the resuspension and cells were incubated
at 37°C with 5% CO2.
2.2. Karyotype Analysis
Karyotype analysis of primary-culture vocal fold fibro-
blasts of passages one through five (P1-P5) and ten (P10)
and grown to 60% confluence was conducted at the Veri-
path Laboratories of UT Southwestern Medical Center
(Dallas, TX). To induce cell cycle arrest and obtain suf-
ficient numbers of cells in metaphase, colcemid (Roche
Laboratories, Nutley, NJ) was added (50 ng/ml final
concentration) overnight for 12-16 hours prior to harvest.
After trypsinization and centrifugation (1500 rpm for 5
min) along with subsequent aspiration of the supernatant,
cells were treated with a hypotonic solution (75 mM KCl)
for 25-30 min at 37°C to promote swelling and to allow
for proper spreading and clear microscopic examination
of the chromosomes.
Cells were fixed using a 3:1 mixture of methanol to
glacial acetic acid (one ml volume) and mixed thor-
oughly prior to centrifugation (1500 rpm for 5 min). Af-
ter a second fixation step using a larger volume of fixa-
tive (7 ml), cells were dropped onto microscope slides in
an incubation chamber (Thermotron, Holland, MI) with
constant humidity for proper spreading of the chromo-
somes. To allow for adsorption of chromosomes onto the
glass surface, slides were incubated at 90°C for 30 min
prior to G-banding by trypsin using Giemsa (GTG) stain.
For the G-banding procedure, heat-treated slides were
immersed in trypsin (1:50 dilution with Isoton II buffer
(Curtis Matheson Scientific, Houston, TX) for 1-2 min
to remove extra chromosomal debris, then in Isoton II
twice to remove trypsin. Two successive washes with
75% and 95% ethanol removed remnants of Isoton II
and a subsequent wash in Gurr buffer eliminated any
ethanol residue. Slides were stained (for 1-2 min) using
Wright’s solution (eosin methylene blue), rinsed with
deionized water and dried with compressed air.
Images of G-banded chromosomes (or karyotypes)
were acquired using the Case Data Manager Version
5.5.2.2 software (Applied Spectral Imaging, Vista, CA),
which arranged chromosomes in a pairwise fashion.
Karyotypes were generated for passages 1-5 and 10 us-
ing 20 metaphase cells. By using the standard number of
20 cells counted, mosaicism of greater than 14% fre-
quency may be excluded at the 95% confidence level
(i.e., p<0.05) [13]. To ensure the reliability and validity
of the analysis, images were analyzed by four individu-
als certified in cytogenetic analysis and the karyotypes
were documented.

G. M. Adams et al. / J. Biomedical Science and Engineering 3 (2010) 148-159
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Table 1. Primer sequences and polymerase chain reaction parameters.
Target Primer Sequence RNA (ng) AT (°C) bp
GAPDH (control) F: GGTGTGAACCATGAGAAG
R: CTGTTGAAGTCAGAGGAGAC 100 50 464
HA Receptor (CD44) F: GAAAGGAGCAGCACTTCAGG
R: CACTTGGCTTTCTGTCCTC 100 50 397
Decorin F: GATGCAGCTAGCCTGAAAGG
R: TCACACCCGAATAAGAAGCC 100 50 274
MMP-1 (Collagenase) F: CAACTTACATCGTGTTGCGG
R: ACCGGACTTCATCTCTGTCG 100 50 395
GAPDH (control) F: ACCCAGAAGACTGTGGATGG
R: TGAGCTTGACAAAGTGGTCG 100 56 382
Procollagen I F: GTTGGTGCTAAGGGTGAAGC
R: ACCGGACTTCATCTCTGTCG 100 56 294
Fibronectin F: TACCAACCTACGGATGACTCG
R: CATCATCGTAACACGTTGCC 100 56 300
GAPDH (control) F: AAGGTCGGAGTCAACGGATTTGGT
R: AGTGATGGCATGGACTGTGGTCAT 100 60 534
HA-synthase 2 F: TACCAACCTACGGATGACTCG
R: CATCATCGTAACACGTTGCC 100 60 403
Collagen I (2) F: TACCCTGGCAATATTGGTCCCGTT
R: GGCAGACCTTGCAATCCATTGTGT 100 60 248
Collagen III (1) F: CTTGATGTGCAGCTGGCATTCCTT
R: AGTCATTGCTCTGCAGATGGGCTA 100 60 686
Hyaluronidase I F: ACTGCAGCAATCACAAAGGCTGAC
R: TATTTGCTGGACTGGTGCCTCTCA 100 60 294
Hyaluronidase II F: AACGTGTGGAGCACTACATTCGGA
R: GAACTGCTGTGCTGCGAACTCAAA 100 60 215
Hyaluronidase III F: TTACAGTCAACCCTCCCAAGCACA
R: TTAGCACTTTACCGACCTTCGCCA 100 60 280
Lysyl Oxidase F: ATGATCACAGGGTGCTGCTCAGAT
R: GTGTGCAGTACATGCAAATCGCCT 100 60 260
GAPDH (control) F: TGGCAAATTCCATGGCACCGTCAA
R: TTGACAAAGTGGTCGTTGAGGGCA 100 62 768
Fibromodulin F: CACTTTCCTCCACAGATGCC
R: GCTGCTTTCTCAATCCAGCC 100 62 387
MMP-12 (macrophage elastase) F: CTGCTGATGACATACGTGGC
R: GTTTGGGATAACCAGGGTCC 100 62 498
AT=annealing temperature; bp=product base pairs; F=forward; R=reverse;
GAPDH=glyceraldehyde 3-phosphate dehydrogenase; MMP=matrix metalloproteinase
Figure 1. An example of PCR agarose gel, showing PCR products gener-
ated from RNA extracted from primary-culture human vocal fold fibro-
blasts of passages 1-5 (P1-P5) and passage 10 (P10) for the hyaluronidase I
target primer as shown in Table 1 (MW=100 base pair DNA molecular
weight marker, Amresco, Solon, OH).
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2.3. Measurement of Population Doubling Time
In order to examine the rate of proliferation of the pri-
mary vocal fold fibroblasts, the cumulative doubling of
the cells was measured by the population doubling time
(PDT). PDT is the time interval required for dividing
cells to double in number [14]. To calculate PDT, a fixed
number (1×104 cells) of vocal fold fibroblasts of a cer-
tain passage (passages 2 to 10) were seeded in each well
of a 6-well plate. After 72 hours, the cells from each well
were collected and counted with a hemocytometer, with
the number of cells designated as NT. Assuming that the
number of cells increases exponentially, at the end of n
generations; each single cell would yield 2n cells. Thus,
after 72 hours, 1×104 seeded cells would produce NT =
(104×2n) cells at harvest. The number of generations (n)
equals to 3.32 (log NT – log 104). By definition, PDT is
the total time elapsed divided by the number of genera-
tions. PDT of the corresponding passage can be obtained
by the following equation [14]:
PDT=72/3.32 (log NT – log 104)
2.4. Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR)
Total RNA was extracted with TRIzol reagent (2 ml)
(Invitrogen, Carlsbad, CA) using a modification of the
method of Chomczynski and Sacchi [15] from fibro-
blasts grown to 80% confluence (~2x106 cells) in two
175 cm2 flasks, for each of passages 1-5 and 10.
All primers for PCR analysis were obtained through
Invitrogen (Carlsbad, CA). Primers for specific targets
and the standard control, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), are listed in Table 1 along
with annealing temperature (AT), product size in base
pairs (bp), and the source of the primer sequences.
Primer sequences for those targets designed in the pre-
sent study were based on sequences published by the
NIH GenBank database, using the SciTools software of
Integrated DNA Technologies (Coralville, IA). Targets
were chosen based on their contributions to and demon-
strated roles in the maintenance of the vocal fold ex-
tracellular matrix [1,16,17]. Furthermore, analyses of
collagenase (MMP-1), macrophage elastase (MMP-12)
and the three isoforms of hyaluronidases (I, II, and III)
were analyzed for conversion of fibroblasts from a ma-
trix-synthesizing to a matrix-degrading phenotype. This
transition serves as a hallmark indicator of the process of
replicative senescence [5].
A master stock (1mM) of each primer was used to
generate a working stock (10 M) for each PCR reaction.
All reactions were carried out using the OneStep RT-
PCR kit (Qiagen, Valencia, CA) coupling the cDNA
synthesis and PCR steps. For each reaction (12.5 l total
volume), the following components were added, with the
final concentrations or amounts noted in brackets: target
forward and reverse primers [400-600 nM], GAPDH
forward and reverse primers [600nM-1500 nM], 1×
RT-PCR buffer (including MgCl2 [12.5 mM]), nucleo-
tides [400 M of each], template RNA (100 ng), RNa-
sin® RNAse inhibitor [7 U] (Promega, Houston, TX) and
the OneStep RT-PCR Enzyme Mix [1:25] (Qiagen, Va-
lencia, CA). Four pairs of primers for the GAPDH con-
trol were used to match four different target annealing
temperatures (Table 1).
Reactions were incubated using a Mastercycler per-
sonal thermal cycler (Eppendorf, Westbury, NY) using a
protocol with an initial denaturation (94°C for 3 min)
and 29-37 cycles of the following steps: denaturation
(94°C for 30 sec), annealing (50°C , 56°C, 60°C or 62°C
for 30 sec) and elongation (72°C for 1 min). As a final
step, PCR products were extended for 7 min at 72°C.
PCR products were electrophoresed in 0.5×TAE buffer
on 2% agarose gels and visualized for densitometric
analysis using a FluorChem HD2 gel documentation
system (Alpha Innotech, San Leandro, CA). An example
of an agarose gel containing PCR products for the hya-
luronidase I target primer is shown in Figure 1. To ver-
ify that the optical density of the PCR products was in
the linear range, three reactions, each with RNA purified
from a different passage, were loaded using two different
volumes (3 and 5 l). PCR products for each target and
for GAPDH were compared using densitometric analysis
with the gel documentation system. To indicate the rela-
tive mRNA expression for each target gene, the densi-
tometric ratio of the target relative to GAPDH was plot-
ted as a function of the passage number.
2.5. Statistical Analysis
For the population doubling time data, one-way analysis
of variance (ANOVA) was conducted to determine if the
proliferation rates of the vocal fold fibroblasts in differ-
ent passages were significantly different from one an-
other. The level of significance (alpha) was set at 0.01.
For the densitometric results of mRNA expressions ob-
tained from RT-PCR, one-way ANOVA was also per-
formed across all passages for each given target. If the
ANOVA results reached statistical significance (p<0.01),
post-hoc analysis was done using the Tukey honestly
significant difference (HSD) test for all possible pair-
wise comparisons of passages (e.g., P1-P2, P3-P4,
P5-P10, etc.). Those pairwise comparisons that contrib-
ute to an overall trend of increase or decrease in the
mRNA expressions of a particular target were deemed
particularly important for the purpose of examining the
effect of passaging or replicative senescence. Also, the
results of the P5-P10 comparisons are shown separate to
the other pairwise comparisons, in order to highlight the
effect of replicative senescence in late passages.

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Figure 2. Passage 1 (P1) karyotype: Systematic ar-
ray (karyotype) of G-banded chromosomes from P1
visualized by Giemsa stain showing an allotment of
23 pairs of chromosomes including the sex chromo-
somes, with three copies of chromosome 3 observed
in 3 of 20 cells examined (boxed area) (only arrays
with chromosomal aberrations are shown).
Figure 3. Passage 2 (P2) karyotype: An array show-
ing three copies of chromosome 3 (boxed area).
Karyotypically normal according to Mitelman [18].
3. RESULTS
3.1. Characterization of Vocal Fold Fibroblast
Karyotype
Chromosomal analysis indicated the presence of an ad-
ditional cell line (noted in 3 of 20 metaphase cells ana-
lyzed) in passage 1 (P1) with three copies of chromo-
some 3 (47, XX, +3[3]/46, XX [17]) (Figure 2, boxed
area). The same anomaly was also found in both passage
2 (P2) and passage 3 (P3), revealing a degree of pse-
udo-mosaicism in 5% of the cells karyotyped (Figures 3
and 4, boxed areas). The term “pseudo-mosaicism” can
be used because the number of cells (one) displaying the
+3 karyotype fell below the minimum number of cells
(two) to be considered a separate clone [18]. The finding
of trisomy 3 can then be considered as statistically insig-
nificant, i.e., a “random gain”. Hence, P2 and P3 are
considered to be karyotypically normal based on this
criterion. For passage 4 (P4), trisomy of chromosome 3
similar to that of P1 was observed in 3 of 20 cells, while
the other cells displayed a normal female karyotype
(Figure 5, boxed area).
Figure 4. Passage 3 (P3) karyotype: As in P2, three
copies of chromosome 3 were found (boxed area).
Karyotypically normal according to Mitelman [18].
Figure 5. Passage 4 (P4) karyotype: Hyperploidy
in P4 as indicated by three copies of chromosome 3
(boxed area).
For passage 5 (P5), three distinct cell lines were ap-
parent. In 3 of 20 cells, three copies of chromosome 3
were observed (Figure 6A, boxed area), along with two
cells showing trisomy of both chromosomes 3 and 8
(Figure 6B, boxed and circled areas, respectively).
Hence, the karyotype for P5 was 47, XX, +3[3]/48, XX,
+3+8[2]/46, XX [15]. For passage 10 (P10), 4 of 20 cells
exhibited the following chromosomal aberrations: trans-
location occurring as a two-break rearrangement; break-
age and reunion at bands 13 and 13.1 of the short arms
(p) of chromosomes 1 and 19, respectively (Figure 7A,
boxed area). The regions distal to these bands have been
swapped. In addition, one of the sex chromosomes has
been deleted, yielding the following karyotype: 45, X,
-X, t(1, 19)(p13, p13.1) [cp4]/46, XX [16]. Trisomy 8
(Figure 7B, circled area) was not listed in the karyotype,
as it was considered a “random gain” [18]. Results for
the cytogenetic analysis are summarized in Table 2.
3.2. Population Doubling Time
Figure 8 shows the mean cumulative population doubl-
ing time (PDT) of the vocal fold fibroblasts as a function
of sub-culturing. The cumulative PDT was seen to in-
crease across different passages, from approximately 30
hours in P2 to over 600 hours for P10. An especially
sharp rise in PDT was associated with the transition

G. M. Adams et al. / J. Biomedical Science and Engineering 3 (2010) 148-159
153
Figure 7. Passage 10 (P10) karyotype: (A) A transloca-
tion has occurred as a two break rearrangement. The re-
gion distal to band 13 on the short arm of chromosome
1 has attached to band 13.1 of chromosome 19 (translo-
cation portion indicated in the boxed area). There was
also the loss of one sex chromosome; (B) One cell out
of four also displayed three copies of chromosome 8
(circled area) and was considered a “random gain”.
Figure 6. Passage 5 (P5) karyotype: (A) Triploidy of
chromosome 3 as seen in P1-P4 (boxed area); (B) An
additional clone was detected displaying +8 abnor-
mality (circled area).
Table 2. Results of cytogenetic analysis.
Passage number Cells with normal diploid karyotype / Total no. of cells Karyotypea
1 17 / 20 47, XX, +3[3] / 46, XX[17]
2 19 / 20 47, XX, +3[1] / 46, XX[19]
3 19 / 20 47, XX, +3[1] / 46, XX[19]
4 17 / 20 47, XX, +3[3] / 46, XX[17]
5 15 / 20 47, XX, +3[3] / 48, XX, +3+8[2] / 46, XX[15]b
10 16 / 20 45, X, -X, t(1;19)(p13;p13.1) [cp4] / 46, XX[16]c
aThe normal diploid clone was listed at the end of the karyotype, along with square brackets [ ], indicating the absolute number of cells in each
clone [18]. bAbnormal clones were listed according to their size with the greatest absolute number first [18]. cThe designation “cp” for composite
karyotype was used because 1 out of 4 cells displayed an additional copy of chromosome 8 (+8) as seen in Figure 7B. As a result, the four cells
represented a composite of three aberrations. Trisomy 8 was not listed in the karyotype as it was considered a “random gain”.
from P7 to P8, suggesting a significant slowing down of
cellular proliferation. Results of single-factor ANOVA
indicated that the differences in PDT across the passages
were statistically significant (F (8, 18)=4.8450, p<0.005).
These findings suggested that the vocal fold fibroblasts
have undergone the process of replicative senescence,
since it has been shown that replicative senescence is
partially signified by an increase in PDT [19].
3.3. RT-PCR
The changes in mRNA expressions of the select target
genes across different passages are shown as densi-
tometric ratios of the targets versus the standard control,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in
Figure 9. Table 3 summarizes the results of statistical
analysis on the dependence of these mRNA expressions
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Figure 8. Cumulative population doubling time (PDT) of pri-
mary vocal fold fibroblasts as a function of passaging (means ±
standard error of the mean; n=3).
on passaging. Pairwise comparisons of passages were
analyzed to detect any possible trends over the passages.
P5-P10 differences are listed separately for targets that are
consistent with induction of senescence. First, for ma-
trix-degrading enzymes, the transcript levels of MMP-1
(collagenase) showed significant increases from the early
passages to passage 10, e.g., a three-fold increase from P5
to P10 (Figure 9A) (p<0.01). For MMP-12 (macrophage
elastase), there was a gradual and significant increase in
its mRNA expressions from P2 to P5 (p<0.01), and then a
dramatic, significant decrease to P10 (p<0.01) (Figure
9B). Both MMP-1 and MMP-12 are implicated in the
remodeling process of the vocal fold ECM, and the
upregulation of MMP-12 has been closely associated with
aging [20]. Hyaluronidase I showed a considerable
upregulation from the early passages to P10, particularly
from P5 to P10 (p<0.01) (Figure 9C). The levels of hya-
luronidase II showed a significant increase from the early
passages to P5, and then a significant decrease to P10
(both at the p<0.01 level) (Figure 9D). For the cross-
linking enzyme lysyl oxidase, significant increases in
mRNA levels were observed with passaging from P3 to
P5, and from P4 to P10 (p<0.01). The increases were ap-
proximately two-fold (Figure 9E).
For procollagen I, the precursor to the a1 peptide of
collagen I, there was a pronounced decrease in mRNA
levels across the passages (p<0.01) (Figure 9F). For
collagen III, the transcript levels of collagen III1 de-
creased significantly across the passages (p<0.01) (Fig-
ure 9G). For fibromodulin (a proteoglycan important for
the structural organization of collagen), there was an
overall reduction in its mRNA levels across the passages,
particularly from P1 to P3 and then from P5 to P10,
where the decrease was 17-fold (p<0.01) (Figure 9H).
For decorin, also important for collagen organization,
Table 3. Results of ANOVA and post-hoc pairwise comparisons of mRNA expressions of target genes across different passages.
A N O V A Post-hoc Tukey HSD Test
Target F (5, 12) p Significant differences for
the following comparisons
Significant differences
for P5-P10 ?
MMP-1 (Collagenase) 218.1 2.4 x 10-11 P5-P10 Yes
MMP-12 (macrophage elastase)10.3 5.14 x 10-4 P2-P5, P4-P10, P5-P10 Yes
Hyaluronidase Type I 9.5 7.4 x 10-4 P5-P10 Yes
Hyaluronidase Type II 196.5 4.5 x 10-11 P1-P5, P2-P5,
P3-P5, P3-P10, P4-P5, P5-P10 Yes
Lysyl Oxidase 101.6 2.15 x 10-9
P1-P2, P1-P3, P1-P4,
P2-P3, P2-P4, P2-P5, P2-P10,
P3-P10, P4-P5, P4-P10
No
Procollagen I 126.1 6.1 x 10-10
P1-P3, P1-P4, P1-P5, P1-P10,
P2-P3, P2-P4, P2-P5, P2-P10,
P3-P5, P3-P10, P4-P5, P4-P10
No
Collagen III (1) 848.9 7.3 x 10-15
P2-P3, P2-P4, P2-P5, P2-P10,
P3-P4, P3-P5, P3-P10,
P4-P5, P4-P10, P5-P10
Yes
Fibromodulin 126.2 6.1 x 10-10
P1-P2, P1-P3, P1-P4, P1-P5,
P1-P10, P2-P3, P2-P10,
P3-P5, P3-P10, P5-P10
Yes
Decorin 30.2 2.12 x 10-6 P1-P5, P1-P10, P2-P4, P2-P5,
P2-P10, P3-P10, P4-P10 No
HA-synthase 2 15.16 7.92 x 10-5 P1-P3, P1-P5, P1-P10 No
Collagen I (2) 1.7 0.21
HA Receptor (CD44) 2.1 0.137
Fibronectin 2 0.149
Hyaluronidase Type III 1.5 0.264
not applicable not applicable

G. M. Adams et al. / J. Biomedical Science and Engineering 3 (2010) 148-159
155
Figure 9. Densitometric ratios for select extracellular matrix target genes (means ± standard deviations; n=3), showing the in-
tegrated density volumes (IDV) or cumulative grey scale values of PCR agarose gel bands for each target relative to those of
the standard control (GAPDH): (A) MMP-1* (collagenase), (B) MMP-12* (macrophage elastase), (C) hyaluronidase I*, (D)
hyaluronidase II*, (E) lysyl oxidase*, (F) procollagen I*, (G) collagen III*, (H) fibromodulin*, (I) decorin*, (J) HA-synthase 2*,
(K) collagen I, (L) CD44, (M) fibronectin, and (N) hyaluronidase III (*p<0.01).
JBiSE

G. M. Adams et al. / J. Biomedical Science and Engineering 3 (2010) 148-159
156
JBiSE
there was a significant decrease in its mRNA levels
across the passages. For instance, the overall reduction
from P1 to P10 was approximately three-fold (p<0.01)
(Figure 9I). For the matrix-synthesizing enzyme HA-
synthase 2, there was a significant overall trend of de-
crease from P1 to P10 (p<0.01), despite an increase from
P3 to P4. In particular, significant differences were
found from the early passages to P10 (Figure 9J).
For the following target genes, no statistically signifi-
cant changes in mRNA expressions were detected as a
function of passaging (p>0.01): collagen I2 (Figure
9K), the HA receptor CD44 (Figure 9L), fibronectin
(Figure 9M) and hyaluronidase III (Figure 9N).
4. DISCUSSIONS
One objective of this study was to establish the onset and
extent of gross chromosomal aberrations in human vocal
fold fibroblasts associated with in vitro aging, or replica-
tive senescence. Chromosomal aberrations were docu-
mented by karyotyping as a function of sub-culturing, or
passaging. Next, alterations in the mRNA expressions
for some key matrix proteins at the transcript level were
observed for the vocal fold fibroblasts across passages,
in order to characterize the consequence of the chromo-
somal aberrations due to aging in vitro.
From the cytogenetic analysis, the +3 karyotype ob-
served in the first four passages (P1-P4) should not be
considered as a significant chromosomal aberration, par-
ticularly for P2 and P3, where the fibroblasts were clas-
sified as karyotypically normal according to ISCN stan-
dards [18]. Beyond passage 4, however, it was apparent
that an additional cell line has appeared with the karyo-
type (+3, +8) which conceivably could be the result of a
nondisjunction with mother cell with a +3 karyotype.
Aberrations observed in passage 5 in this study were
comparable to those of passage 4 in Thibeault et al. [7].
Yet the severity of the aberrations in this study was rela-
tively modest, e.g., trisomy 8 in 3 cells of 20 in P4 in
this study versus the numerous anomalies in P4 of
Thibeault et al. [7], including the loss of sex chromo-
some in 6 of 21 cells, translocation in 3 of 21 cells; and
rearrangement involving chromosomes 2, 5, 10, 14, 15.
This disparity in the number and severity of chromoso-
mal abnormalities was likely due to the difference in age
of the donors of the studies (58 years old in this study
versus 78 years old in Thibeault et al. [7]). The suscepti-
bility of vocal fold fibroblasts to these aberrations ap-
parently increased with age and should be targeted in
future studies.
Based on the RT-PCR analysis for those target genes
demonstrating statistically significant differences across
different passages, our data on the changes in mRNA
expressions of the targets can be divided roughly into
two categories: 1) target genes involving matrix-syn-
thesizing enzymes and matrix-degrading enzymes, in-
cluding MMP-1, MMP-12, hyaluronidases, and HA-
synthase 2; and 2) targets contributing to the regulation
of the ECM organization, including procollagen I, col-
lagen III, decorin, fibromodulin, and lysyl oxidase.
For target genes involving matrix-synthesizing and
matrix-degrading enzymes, it has been considered that
the shift of fibroblasts from a matrix-synthesizing to a
matrix-degrading phenotype is one of the hallmark indi-
cators for replicative senescence [5]. This notion was
supported by numerous studies that showed upregulation
of matrix metalloproteinases (MMPs) in senescent fibro-
blasts [21,22,23]. Such a shift towards a matrix-degrading
phenotype was demonstrated in the present study by the
modulations observed in the following targets.
The upregulation of both MMP-1 (collagenase) and
MMP-12 (macrophage elastase) with passaging of human
vocal fold fibroblasts is consistent with in vitro aging
seen in other kinds of fibroblasts, such as dermal fibro-
blasts [24,25,26]. Hyaluronidases are responsible for the
cleavage of hyaluronic acid (HA). Type I and type II
hyaluronidases are the most common isoforms expressed
in human somatic tissues [27]. HA, a highly polarized
long-chain glycosaminoglycans molecule, is abundantly
found in the vocal fold lamina propria [28]. It is critical
to the regulation of tissue hydration and is important for
the maintenance of an optimal viscoelasticity of the lam-
ina propria for vocal fold vibration [2,4,28]. From the
PCR analysis, the upregulation of hyaluronidase II from
P1 to P5 and the upregulation of hyaluronidase I from P5
to P10 were consistent with a proposed model for their
concerted action in the degradation of HA [28]. Specifi-
cally, hyaluronidase II cleaves HA first into 20 kDa
fragments, followed by hyaluronidase I digesting them
into smaller fragments [29]. Accumulation of the 20 kDa
fragments leads to an increase in transcripts of hyalu-
ronidase I by mass action, as seen in P10. The actions of
these enzymes in concert were consistent with a shift
towards a matrix-degrading phenotype.
Mammalian HA synthesis is accomplished in part by
HA-synthase 2 [16]. The levels of HA-synthase 2 showed
a steady decrease from P1 to P3, and remained at low
levels for P5 and P10, indicating that the synthesis of
HA has been reduced in these fibroblasts. This finding
further supported our argument that across the passages,
vocal fold fibroblasts have shifted from a matrix-synthesiz-
ing to a matrix-degrading phenotype, consistent with
replicative senescence.
The changes in mRNA expressions of the remaining
targets strongly implicated that the in vitro aging of these
fibroblasts would lead to a disorganized and structurally
compromised lamina propria ECM. First, for collagen
III 1, which is a major structural fibrous protein of the
vocal fold lamina propria and macula flava [30,31], there
was a steady and significant decrease in the transcript
level with passaging. Type III collagen fibers were found

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to be thicker than type I fibers, with their interactions
likely contributing to the viscoelasticity of the lamina
propria and the tensile strength of the macula flava nec-
essary for bearing the mechanical stresses of vocal fold
vibration [32]. For procollagen I, a significant decrease
in its mRNA levels was also seen with passaging. This
downregulation did not correspond to a significant de-
crease in collagen I mRNA levels, because different pep-
tides were coded for the two targets (1 for procollagen
I and 2 for collagen I). Our findings of collagen III,
collagen I and procollagen I expressions suggested a
lower turnover rate for collagen, leading to an ECM in-
creasingly cross-linked and difficult to degrade, as ob-
served in aged human vocal folds [33].
Decorin contributes to regulate the organization and
overall size of collagen fibrils via its glycosaminogly-
cans moiety [34], presumably reducing collagen fiber
size and density [35]. Lower levels of decorin have been
associated with disorganized collagenous supramolecu-
lar assemblies in scar tissue [36]. The observed decrease
in the transcript levels of decorin along with the decrease
in collagen III suggested that the capability of fibroblasts
for facilitating structural ECM organization has been
impaired due to aging in vitro.
Fibromodulin is a small proteoglycan that facilitates
the construction of other proteins such as collagens I and
II into fibrillar structures [17]. It has been shown that
fibromodulin-knockout mice have disorganized collagen
fibrils in their Achilles tendons, with reduced overall
cross-sectional area when compared to normal control
[37]. Our results showed a dramatic, significant 17-fold
decrease in the level of fibromodulin from P5 to P10.
Combined with the decorin data, lower fibromodulin
levels supported the notion that the capability of the fi-
broblasts for ECM protein organization is compromised
with passaging.
The cross-linking enzyme lysyl oxidase catalyzes the
removal of amino groups from certain hydroxylysine
and lysine residues in collagen, cross-linking one colla-
gen molecule to another [38]. Lysyl oxidase is appar-
ently the only known enzyme responsible for cross-
linking elastin monomers [39]. Such cross-linking would
likely increase the elastic modulus for both collagen and
elastin fibers. Increases in tissue stiffness of the vocal
fold lamina propria and the macula flava have been seen
with aging [17,37], which could be related to the in-
creased cross-linking of collagen and elastin fibers. Our
data showed a clear upregulation in lysyl oxidase from
P3 to P10, consistent with this modulation in vocal fold
tissue stiffness with aging.
These results, along with the dramatic decrease in
macrophage elastase (MMP-12) from P5 to P10, sug-
gested that the gene expressions of senescent vocal fold
fibroblasts in vitro are consistent with a proposed model
for in vivo aging of the vocal fold. This model suggests
that as vocal folds age, elastin fibers accumulate with a
prolonged half-life due to lower elastase levels and un-
dergo extensive cross-linking via lysyl oxidase, contrib-
uting to increased tensile strength and tissue stiffness.
The increase in lysyl oxidase levels with passaging in
our study contradicted the results for aged rats in Ding
and Gray [39], but may nonetheless reflect inherent dif-
ferences between the species, and between in vitro and
in vivo models.
Overall, based on our data on cytogenetic analysis,
PDT and mRNA expressions of target genes, we have
established the sub-culturing limit beyond which replica-
tive senescence was observed in primary-culture human
vocal fold fibroblasts. This limit of 5 passages was critical
for establishing representative in vitro models for tissue
engineering approaches involving the use of primary-
culture fibroblasts. For example, primary vocal fold fibro-
blasts seeded in decellularized ECM scaffolds have been
shown to be promising for vocal fold regeneration [8].
The therapeutic potential of injection of autologous fibro-
blasts as well as the incorporation of growth factors or
biomaterial implants with fibroblasts for treating vocal
fold scarring has also been targeted [9,10,40]. In those
studies, autologous fibroblasts were harvested by biopsy
and sub-cultured for up to the sixth passage, for up to 30
days. The current findings suggested that some of the fi-
broblasts in those studies may have become senescent,
compromising their findings and conclusions.
Nonetheless, it should be noted that the results of this
study should be considered preliminary, based on the
harvest of vocal fold fibroblasts from only a single indi-
vidual. Also, the vocal fold fibroblasts were only cul-
tured statically, unlike in physiological situations when
they are subjected to a variety of mechanical stresses,
particularly vibratory stresses. Titze et al. showed that
axial and vibratory strains applied to human vocal fold
fibroblasts cultured on a polymeric substrate resulted in
significant changes in mRNA expressions of a number of
matrix proteins [9]. Further studies involving bioreactors
capable of simulating the micromechanical environment
in vivo are required to address this important issue. In
addition, the measurement of gene expressions at the
transcript level does not always directly reflect expres-
sions of the corresponding proteins. Changes in mRNA
expression of a certain protein would depend on the
half-life of that protein, and on its regulation pathways.
Future studies should therefore involve the measurement
of protein levels with methods such as enzyme-linked
immunosorbent assays.
5. CONCLUSIONS
In summary, the patterns of chromosomal abnormalities
and mRNA expressions of matrix proteins across differ-
ent passages of primary-culture vocal fold fibroblasts
indicated that the fibroblasts underwent replicative se-

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nescence, with the onset of senescence not occurring
before the fifth passage. These preliminary data sug-
gested that an acceptable limit of sub-culturing may be
established to facilitate the development of representa-
tive in vitro models of the vocal fold ECM, allowing for
the evaluation of a variety of tissue engineering ap-
proaches for vocal fold reconstruction.
6. ACKNOWLEDGEMENTS
This study was supported by the National Institutes of Health, NIDCD
Grant No. R01 DC006101. The authors would like to thank Dr. J.T.
Hsieh for the use of his gel documentation system, and Glendon
Plumton for his assistance in the RT-PCR work.
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