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Age Dependence of Cellular Properties of Human Septal Cartilage
Implications for Tissue Engineering
Nicole Rotter, MD;
Lawrence J. Bonassar, PhD;
Geoffrey Tobias, MD;
Martin Lebl, MD;
Amit K. Roy, PhD;
Charles A. Vacanti, MD
Arch Otolaryngol Head Neck Surg. 2001;127:1248-1252.
ABSTRACT
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Background The persistent need for cartilage replacement material in head and neck
surgery has led to novel cell culture methods developed to engineer cartilage.
Currently, there is no consensus on an optimal source of cells for these endeavors.
Objectives To evaluate human nasal cartilage as a potential source of chondrocytes
and to determine the effect of donor age on cellular and proliferation characteristics.
Subjects Nasal cartilage specimens were obtained after reconstructive surgery
from 46 patients ranging in age from 15 to 60 years.
Methods Specimens were weighed and chondrocytes were isolated by digestion in
0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures
until confluency, then seeded onto polylactic acid–polyglycolic acid
scaffolds. Seeding efficency was determined by quantification of DNA content
of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell
yields, cell content, and doubling time were also measured and correlated
to donor age.
Results Mean (±SD) cartilage mass obtained (648 ± 229 mg) is higher
than from typical biopsy specimens of auricular cartilage, and the cellular
characteristics show a higher proliferation rate than auricular chondrocytes.
Cell yield increased with age, while doubling time decreased with age in samples
from patients ranging from 15 to 60 years old.
Conclusions The use of nasal septal cartilage as a source of cells for tissue engineering
may be valid over a wide range of patient ages. The large tissue yield and
consequent cell yield make this tissue a potential starting source of chondrocytes
for large-volume tissue-engineered implants.
INTRODUCTION
TISSUE ENGINEERING has recently become a viable method for generating
material for the replacement of cartilage defects in the head and neck. It
has been demonstrated that different types of chondrocytes are capable of
forming cartilage specimens in vitro and in vivo with the help of resorbable
polymer scaffolds. Several studies have documented the use of bovine articular
chondrocytes1-4
as well as porcine5 and canine6
auricular cells in the generation of tissue-engineered cartilage. The translation
of this technology to clinical applications necessitates that human cells
be characterized as thoroughly to determine their suitability for applications
of tissue engineering. Human septal chondrocytes have also been demonstrated
to be capable of forming cartilage specimens, closely resembling the native
cartilage from which they were derived.7-9
However, the optimal source of tissue for harvest of cartilage still needs
to be determined.
Little is known about the specific characteristics of different cartilage
types for tissue engineering.10 This information
is of critical importance in the effort to translate tissue engineering into
clinical practice. The density of cells in the tissue, as well as the efficiency
with which they are isolated and the in vitro growth rates, are important
design constraints for planning the size of tissue needed for harvest and
the time necessary for cell expansion. Furthermore, it is still unknown whether
these characteristics and others such as cell-scaffold interactions are dependent
on patient age.
Studies investigating growth characteristics of the human septum in
the context of midfacial development11-12
report an increasing or constant number of chondrocytes in the human nasal
septum in dependency of the location within the septum. A recent study using
histochemical staining, however, demonstrated a decreasing cell number with
increasing donor age, although it did not take specific locations into account.13 A decrease in the cellularity is believed to occur
in articular cartilage, although it remains controversial whether these observations
are part of the physiologic aging process or result from beginning osteoarthritic
changes.
To investigate human septal cartilage as a potential source for tissue
harvest for tissue engineering applications, the goals of this study were
(1) to describe the cellular characteristics relevant to human septal cartilage
specimens including sample mass, cell density, isolation efficiency, scaffold
seeding efficiency, and growth rate; and (2) to determine the extent to which
these properties depend on the age of the donor.
MATERIALS AND METHODS
HARVEST OF CARTILAGE AND CELL ISOLATION
Human septal cartilage was obtained after reconstructive septorhinoplasty
in accordance with the guidelines of the University of Massachusetts Medical
Center, Worcester, and Englewood Hospital, Englewood, NJ. Donor age ranged
from 15 to 60 years, with a mean (±SD) age of 32.9 ± 12.9 years.
Samples were obtained from 46 patients, 27 female and 19 male. Immediately
after surgery, samples were placed in Dulbecco minimum essential medium (Life
Technologies, Grand Island, NY) containing penicillin G, 100 U/mL; streptomycin,
100 µg/mL; and amphotericin B, 0.25 µg/mL (Life Technologies)
for 4 to 16 hours before processing. Specimens were freed of surrounding perichondrium,
bone, or connective tissue, and the wet weights were determined. A 6-mm punch
biopsy was obtained, weighed, and frozen at -20°C until biochemical
testing. The remaining cartilage sample was exposed to 0.2% collagenase type
II (Worthington, Lakewood, NJ) for 10 to 14 hours to isolate the chondrocytes
as described previously.1
CELL CULTURE
Chondrocytes were seeded at a density of 8000 cells/cm2 and
maintained at 37°C in a 5% carbon dioxide atmosphere in primary monolayer
culture until confluency (approximately 12-14 days). Dulbecco minimum essential
medium supplemented with penicillin G, 100 U/mL, streptomycin, 100 µg/mL,
and amphotericin B, 0.25 µg/mL; ascorbic acid, 25 µg/mL; and 10%
fetal bovine serum served as culture medium, with fresh medium added every
2 to 3 days. Cells were released from culture flasks by treatment with 0.05%
trypsin-EDTA (Life Technologies) and counted with a hemocytometer using trypan
blue exclusion. Chondroytes were seeded onto polyglycolic acid (PGA) (Albany
International, Mansfield, Mass) disks coated with 0.5% polylactic acid (PLA)
(Polysciences Inc, Warrington, Pa) at a density of 30 x 106
cells per milliliter. The disks had a diameter of 10 mm and a thickness of
2 mm. The PLA coating was achieved by immersion of prefabricated PGA disks
into a 0.5% solution of PLA in methylene chloride (Sigma-Aldrich Corp, St
Louis, Mo).14
CELL SEEDING AND DETERMINATION OF SEEDING EFFICIENCY
Seeding was performed by using a pipetting technique, in which a volume
corresponding to the scaffold volume was slowly pipetted onto the prewetted
polymer disks. Cells were allowed to seed for 2 hours before addition of medium.
Twenty-four hours after the initial cell seeding procedure, scaffolds were
digested with papain, 0.125 mg/mL, and digests were assayed for DNA with Hoechst
dye 33258.15 The number of cells in the constructs
was calculated by means of an assumed amount of 7.7 pg of DNA per chondrocyte.15
STATISTICS
Linear regression analysis was used to evaluate the age dependence of
cell density, isolation efficiency, doubling time, and seeding efficiency.
The t test was used to evaluate sex differences in
cell density, isolation efficiency, doubling time, and seeding efficiency.
By means of experimentally determined variances, the powers of age and sex
analyses were greater than 0.8. Unless otherwise indicated, data are given
as mean ± SD.
RESULTS
The average mass of specimens of human nasal cartilage obtained by reconstructive
(septo)rhinoplasty was 648.1 ± 229.4 mg. The mass was not dependent
on donor age or sex (Table 1).
The average mass of samples from male donors was 690.3 ± 249.9 mg,
and from female donors, 614.6 ± 227.6 mg.
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Group and Sex Averages of Cell Culture Variables*
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The average cell yield was 4.45 x 103 ± 2.28
x 103 cells per mg after a 16-hour digestion in 0.2% collagenase
type II. The yield increased significantly (P<.02)
with donor age (Figure 1), with
regression analysis showing a 50% increase in yield from tissue from 60-year-old
patients compared with samples from 15-year-old patients. There was no significant
correlation with sex (Table 1).
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Figure 1. Number of cells harvested by collagenase
digestion per weight of nasal septal cartilage vs donor age for 45 patients.
Values of correlation coefficient (R2)
and significance level (P) were calculated by linear
regression analysis.
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The cell content calculated from measurements of DNA content did not
depend on the donor age (Figure 2)
or donor sex (Table 1). Linear
regression analysis showed a slight (approximately 5%) increase in cell density
by 60 years of age compared with 15 years of age, but this was not statistically
significant (P>.05).
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Figure 2. Cell content as determined by
DNA assay digestion per weight of nasal septal cartilage vs donor age for
45 patients. Values of correlation coefficient (R2) and significance level (P) were calculated
by linear regression analysis.
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Surprisingly, a comparison of cartilage cell content, as determined
by DNA quantification, and cell yield, as determined from cell counts after
collagenase digestion, indicated that there was no positive correlation between
these 2 variables (Figure 3). This
suggests a large variability in the cell yield from collagenase type II digestion.
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Figure 3. Cell content as determined by
DNA assay digestion per weight vs number of cells harvested by collagenase
digestion per weight for 45 patients. Values of correlation coefficient (R2) and significance level (P) were calculated by linear regression analysis.
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Isolation efficiency, calculated as the quotient of the number of enzymatically
isolated cells and the cell content as determined by DNA assay, averaged 11.9%,
reflecting that most of the chondrocytes in the tissue either are not liberated
or do not remain viable after the procedure. Linear regression analysis indicated
that isolation efficiency increased from 10% in tissue from 15-year-old patients
to 14% in tissue from 60-year-old patients (Figure 4), but that this change was not statistically significant
(.05<P<.1). The isolation efficiency did not
vary with donor sex (Table 1).
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Figure 4. Isolation efficiency vs donor
age for 45 patients. Values of correlation coefficient (R2) and significance level (P)
were calculated by linear regression analysis.
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The average doubling time of human septal chondrocytes was 2.6 ±
0.98 days. This variable showed a significant dependence on donor age, with
cells from older patients proliferating more rapidly (P<.02) (Figure 5). Linear
regression analysis indicated that doubling time decreased from more than
3 days for cells from 15-year-old patients to less than 2 days for cells from
60-year-old patients. Proliferation was independent of sex (Table 1).
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Figure 5. Doubling time vs donor age for
22 patients. Values of correlation coefficient (R2) and significance level (P) were calculated
by linear regression analysis.
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The seeding efficiency, calculated as the number of cells that adhered
to the scaffold after 24 hours normalized to the number of cells placed on
the scaffold, averaged 25.1% (Table 1).
The seeding efficiency did not vary with donor age (Figure 6) or sex (Table 1).
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Figure 6. Seeding efficiency (number of
cells attached normalized to number of cells seeded) vs donor age for 18 patients.
Values of correlation coefficient (R2)
and significance level (P) were calculated by linear
regression analysis.
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COMMENT
The goal of this study was to characterize the suitability of human
septal cartilage for tissue engineering on the basis of cellular characteristics.
The results indicated that isolation and cellularity of septal chondrocytes
are independent of age over a large age span. Doubling time decreases with
increasing age, indicating more rapid proliferation, and isolation efficiency
increases slightly. Although significant age trends exist, the data from the
current study suggest that the use of nasal septal cartilage as a source of
cells for cartilage tissue engineering remains viable for all patient ages
sampled.
An important measure in assessing the suitability of a certain type
of tissue for application in tissue engineering is the amount of tissue available
at initial harvest. No consensus exists on which type of cartilage is best
suited for certain applications. Several studies have used bovine articular
cartilage3-4 to demonstrate feasibility,
as it is readily available in large amounts, thereby omitting the problem
of tissue selection and cell amplification. However, for clinical applications
this source is not suitable.
Common locations for cartilage harvests in clinical settings are the
outer ear, the rib, and the nasal septum. The mass of recovered cartilage
(about 650 mg) in our study was large compared with tissue gained by surgery
on the outer ear (about 50 mg).16 This is of
great relevance, as the obtainable volume determines the number of primary
cells that can be used for tissue engineering in vitro. In general, chondrocytes
are amenable to proliferation in monolayer culture. However, after extended
culture and passaging, chonodrocytes lose their specific phenotype and thus
the ability to produce cartilage-specific matrix products.17
It has been shown that redifferentiation of chondrocytes is possible in different
3-dimensional culture systems.18 Nevertheless,
it remains in question whether tissues formed by vastly expanded chondrocytes
will have the same quality and characteristics as tissues from primary or
slightly expanded cells. Therefore, it is important to initially obtain a
sufficient number of cells to avoid extensive expansion in monolayer culture.
Clearly, this requires the harvest of an adequate tissue volume.
The volume of the tissue for harvest should be determined before the
procedure by correlation with the desired volume of the tissue-engineered
construct. To reconstruct large defects like, for example, a whole adult human
ear, the necessary cell number is in the range of 1 x 108
to 5 x 108 cells. The primary aim, therefore, is to obtain
a sufficient amount of tissue, allowing amplification in monolayer culture
for 2 to 3 weeks without extensive cell dedifferentiation. The large variation
in the efficiency of the isolation procedure (Figure 3) suggests that conservative estimates must be used when
procedures are planned for harvest of nasal cartilage for tissue engineering.
However, our data show that this expansion of cell number could take place
entirely in primary culture, which would minimize the risk of dedifferentiation
and loss of chondrocyte cell function.
The doubling time of human nasal septal chondrocytes (2.6 ± 1.0
days) is shorter than that reported for human ear chondrocytes (3.4 days).16 This is of significant concern, since doubling time
would directly limit the time between tissue harvest and reimplanation of
an engineered construct. Given the average cell yield (approximately 12%)
and the doubling time (2.6 days), it would take approximately 8 days of culture
(3 doublings) to obtain the same number of cells present in the original tissue.
Further culture beyond this time would allow for expansion of cell population
beyond that originally harvested (eg, 13 days of total culture time would
provide 4 times the amount of cells originally harvested, or enough to make
approximately 2.5 cm3 of tissue).
While the phenomenon of dedifferentiation limits the possible level
of expansion of the cell population, data from the current study begin to
allow for estimation of the volume of tissue that can ultimately be fabricated
by these techniques. Previous work18 has demonstrated
that placement of cells into scaffolds after second passage preserves chondrocyte
phenotype markers such as proteoglycan and type II collagen production. A
conservative estimate is that each passage will allow for an 8-fold expansion,
such that 2 passages will convey a 64-fold expansion of the cells obtained
with a 12% efficiency from the tissue. The net result is an approximately
7-fold (12%x64) increase in tissue mass, or approximately 4.5 cm3 of implant. This would be of great utility in a variety of procedures
in craniofacial reconstruction.
The slight increase in isolation efficiency as well as the decrease
in doubling time might be explained by changes in cellular characteristics
into a more fibroblastlike cell type. This is consistent with previous reports
that the proteoglycan content of human nasal cartilage decreases with age,
while the collagen content increases.19 Embedded
in a slightly more fibrous tissue matrix, isolation would be facilitated and
the fibroblastlike cell would tend to proliferate more rapidly than hyaline
chondrocytes. If this is true, one may expect decreased matrix synthesis from
cells in these tissues as well, but this has not been established.
The current study did not demonstrate any characteristics to exclude
any patients aged 15 to 60 years from such procedures. The seeding efficiency
was also independent of donor age. However, with the use of the classic pipette
seeding technique, the efficiency with PLA-coated PGA scaffolds was relatively
low, with an average of 25%. Interestingly, this is consistent with data obtained
by seeding of bovine articular chondrocytes on PGA scaffolds, coated with
different concentrations of PLA,20 thus indicating
a comparable behavior of bovine articular and human nasal septal chondrocytes
with regard to seeding characteristics. Other groups have demonstrated seeding
efficiencies of 100% with more complex procedures requiring specialized equipment.4, 21
In summary, septal cartilage appears to be a suitable source for tissue
harvest for engineering of cartilage in vitro. It is easy to obtain with low
donor site morbidity and offers sufficient cell numbers. Many cellular characteristics
seem to be independent of donor age for the span from 15 to 60 years, thus
defining the suitable patient collective. Further studies on the characteristics
of the tissue-engineered cartilage of these specimens are currently being
conducted.
AUTHOR INFORMATION
Accepted for publication May 16, 2001.
This study was supported by a grant from the German Academic Exchange
Society, Munich (Dr Rotter), and the University of Massachusetts Medical School,
Worcester.
Corresponding author and reprints: Charles A. Vacanti, MD, Department
of Anesthesiology, University of Massachusetts Medical School, Worcester,
MA 01655 (e-mail: Charles.Vacanti{at}umassmed.edu).
From the Center for Tissue Engineering, Department of Anesthesiology,
University of Massachusetts Medical School, Worcester (Drs Rotter, Bonassar,
Lebl, Roy, and Vacanti); Department of Otorhinolaryngology, Head and Neck
Surgery, Ludwig-Maximilians-University of Munich, Munich, Germany (Dr Rotter);
and Department of Otolaryngology, Englewood Hospital, Englewood, NJ (Dr Tobias).
REFERENCES
| |
1. Vacanti JP, Morse MA, Saltzman WM, Domb AJ, Perez-Atayde A, Langer R. Selective cell transplantation using bioabsorbable artificial polymers
as matrices. J Pediatr Surg. 1988;23:3-9.
2. Vacanti CA, Langer R, Schloo B, Vacanti JP. Synthetic polymers seeded with chondrocytes provide a template for
new cartilage formation. Plast Reconstr Surg. 1991;88:753-759.
WEB OF SCIENCE
| PUBMED
3. Puelacher WC, Mooney D, Langer R, Upton J, Vacanti JP, Vacanti CA. Design of nasoseptal cartilage replacements synthesized from biodegradable
polymers and chondrocytes. Biomaterials. 1994;15:774-778.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
4. Vunjak-Novakovic G, Obradovic B, Martin I, Bursac PM, Langer R, Freed LE. Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog. 1998;14:193-202.
FULL TEXT
| PUBMED
5. Cao Y, Rodriguez A, Vacanti M, Barra C, Arevalo C, Vacanti CA. Comparative study of the use of poly(glycolic acid), calcium alginate
and pluronics in the engineering of autologous porcine cartilage. J Biomater Sci Polym Ed. 1998;9:475-487.
PUBMED
6. Britt JC, Park SS. Autogenous tissue-engineered cartilage: evaluation as an implant material. Arch Otolaryngol Head Neck Surg. 1998;124:671-677.
FREE FULL TEXT
7. Sittinger M, Bujia J, Rotter N, Reitzel D, Minuth WW, Burmester GR. Tissue engineering and autologous transplant formation: practical approaches
with resorbable biomaterials and new cell culture techniques. Biomaterials. 1996;17:237-242.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
8. Rotter N, Sittinger M, Hammer C, Bujia J, Kastenbauer E. Transplantation of in vitro cultured cartilage materials: characterization
of matrix synthesis [in German]. Laryngorhinootologie. 1997;76:241-247.
PUBMED
9. Rotter N, Aigner J, Naumann A, et al. Cartilage reconstruction in head and neck surgery: comparison of resorbable
polymer scaffolds for tissue engineering of human septal cartilage. J Biomed Mater Res. 1998;42:347-356.
FULL TEXT
| PUBMED
10. Cardenas-Camarena L, Gomez RB, Guerrero MT, Solis M, Guerrerosantos J. Cartilaginous behavior in nasal surgery: a comparative observational
study. Ann Plast Surg. 1998;40:34-38.
PUBMED
11. Vetter U, Pirsig W, Heinze E. Growth activity in human septal cartilage: age-dependent incorporation
of labeled sulfate in different anatomic locations. Plast Reconstr Surg. 1983;71:167-171.
PUBMED
12. Vetter U, Heit W, Heinze E, Pirsig W. Growth of the human septal cartilage: cell density and colony formation
of septal chondrocytes. Laryngoscope. 1984;94:1226-1229.
PUBMED
13. Edelstein DR. Aging of the normal nose in adults. Laryngoscope. 1996;106:1-25.
14. Cao Y, Vacanti JP, Paige KT, Upton J, Vacanti CA. Transplantation of chondrocytes utilizing a polymer-cell construct
to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg. 1997;100:297-302.
WEB OF SCIENCE
| PUBMED
15. Kim YJ, Sah RLY, Doong JYH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem. 1988;174:168-176.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
16. Borland K, Zhou T, Birkhead J, Omstead D. Injectable hydrogels containing autologous chondrocytes as engineering
tissue bulking agents [abstract]. In: Programs and abstracts of the 2nd Annual Meeting of the Bioartificial
Organ Society; July 18-21, 1998; Banff, Alberta. Page 11.
17. von der Mark K, Gauss B, von der Mark H, Muller P. Relationship between shape and type of collagen synthesized as chondrocytes
lose their cartilage phenotype in culture. Nature. 1977;267:531-532.
FULL TEXT
| PUBMED
18. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen
phenotype when cultured in agarose gels. Cell. 1982;30:215-224.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
19. Rotter N, Roy A, Tobias G, et al. Age dependence of biochemical and biomechanical properties of non-articular
hyaline cartilage [abstract]. In: Programs and abstracts of the 47th Annual Meeting of the Orthopaedic
Research Society; February 25-28, 2001; San Francisco, Calif. Page 434.
20. Moran JM, Bonassar LJ. Fabrication and characterization of PLA/PGA composites for cartilage
tissue engineering [abstract]. Tissue Eng. 1998;4:498.
21. Chu CR, Coutts RD, Yoshioka M, Harwood FL, Monosov AZ, Amiel D. Articular cartilage repair using allogeneic perichondrocyte-seeded
biodegradable porous polylactic acid (PLA): a tissue-engineering study. J Biomed Mater Res. 1995;29:1147-1154.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
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