Vol.2, No.6, 609-612 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Chondrocyte viability depends on the preservative
Krzysztof Gawęda1*, Marta Tarczyńska1, Ewa Olender2, Izabela Uhrynowska-Tyszkiewicz2,3,
Artur Kamiński2,3
1Department of Orthopaedics and Traumatology, Medical University of Lublin, Lublin, Poland;
*Corresponding Author: krzylub@o2.pl
2National Centre for Tissue and Cell Banking, Warsaw, Poland
3Department of Transplantology and Central Tissue Bank, Centre of Biostructure Research, Medical University of Warsaw, Warsaw,
Received 11 February 2010; revised 15 March 2010; accepted 18 March 2010.
Fresh osteochondral grafts find broad applica-
tion in the treatment of extensive and focal
damages of joint surfaces. The maintenance of
chondrocyte viability of the collected grafts is of
key importance. Aim: The evaluation of chon-
drocyte survivability in a stable temperature of
40C in different preservative solutions is the aim
of this work. Method: Chondrocyte survivability
has been evaluated in saline solution (group I),
Ringer solution (group II), saline with an addi-
tion of hyaluronic acid (group III) and saline en-
riched with glucosamine sulphate (group IV).
The amount of live chondrocytes was examined
on the day of collection and subsequently after
1, 2, 3,6,12, and 21 days using the Promega MTT
test. Results: The highest number of live chon-
drocytes as calculated for 1g of hyaline carti-
lage after 21 days was ascertained in group IV
(saline with glucosamine sulphate). The lowest
number of live chondral cells was observed in
group II (saline with hyaluronic acid). Chon-
drocyte survivability in saline (group I) was
higher than in the Ringer solution (group II).
Conclusions: The enrichment of saline solution
with glucosamine sulphate protracts the viabil-
ity of chondrocytes in fresh osteochondral gra-
fts prepared for chondral transplantation.
Keywords: Chondrocyte; Preservative Solution;
Osteochondral Grafts; Fresh Grafts
The use of fresh allogeneic osteochondral grafts to repair
extensive damage to joint surfaces spans more than 100
years [1]. Grafts of many different types and sizes have
been used, from various cylindrical osteochondral grafts
with radii ranging from several millimeters to larger than
1 cm, through shell grafts, to the transplantation of
whole joint surfaces of tibial or femoral condyles [2].
The size of fresh allogeneic osteochondral grafts not-
withstanding, the determination and maintenance of safe
conditions for the donor joint surfaces remains an ongo-
ing issue. It is crucial to maintain strict sanitary stan-
dards for grafts until their implantation. Although rules
regarding such standards are determined by laws, and
thus differ by country, it is important for hospitals to
organize a specialized transplant service with procedural
guidelines, which will mitigate difficulties encountered
while performing this repair method. The tasks of such a
service should include recruiting donors and preparing
potential recipients for transplantation. Efficient use of
fresh allogeneic grafts of joint surfaces is essential be-
cause the chondrocytes of transplanted cartilage atrophy
rapidly. Tissue survival can be extended by cooling col-
lected fragments. However, freezing typically destroys
live chondral cells and disintegrates the intercellular
matrix structure [3]. Thus, maintaining the viability of
collected chondrocytes is a key issue to be addressed at
centers that use this method of joint cartilage repair. Sig-
nificant chondrocyte survival can be maintained for only
1-2 weeks by preserving osteochondral grafts in normal
saline with antibiotics, buffered normal saline, or Ringer
solution cooled to 4°C, although this period can be ex-
tended [4-6]. Thus there is a continuing need for meth-
ods that prolong the safe preservation and transport of
graft materials with live chondrocytes.
Pharmaceuticals and dietary supplements that pur-
portedly improve chondrocyte vitality and the ability of
damaged chondral surfaces to self-repair have gained
K. Gawęda et al. / HEALTH 2 (2010) 609-612
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
popularity in recent years. Various derivatives of hyalu-
ronic acid and substances containing glucosamine are
most frequently used.
2. AIM
The widespread use of hyaluronic acid and glucosamine
compounds in the pharmacotherapy of focal and gener-
alized joint cartilage damages raises the prospect of pos-
sibly extending chondrocyte vitality by adding these
substances to the transport solutions for osteochondral
grafts. Therefore, in this study, we determined the sur-
vival of human chondral cells stored in vitro at the same
temperature, but in different conserving solutions.
The distal femoral epiphysis was collected along with
the cartilage coating from six male donors, aged 21-48
years, in the operating room after the conclusion of
multi-organ collections. The obtained osteochondral spe-
cimens were divided into four equal parts. Each was
randomly assigned for immersion in one of the following
four media: normal saline solution (group I), Ringer so-
lution (group II), normal saline solution with 20 mg so-
dium salt of hyaluronic acid (group III), or normal saline
solution enriched with 1.5 g glucosamine sulphate
(group IV). Each medium was enriched with 1.0 g third-
generation cephalosporin. Each of the obtained quarters
of the distal femoral epiphysis was subsequently divided
into seven equal parts, which were placed in separate
sterile containers filled with the evaluated solution. All
samples were stored at 4°C. Live chondrocyte numbers
in the initial samples of each group were quantitatively
analyzed within 24 h after collection using colorimetric
reduction in the Promega MTT (3-(4, 5-dimethylthiazol-
2-yl)2, 5-diphenyl tetrazolium bromide) test. Evaluations
were subsequently conducted on Days 1, 2, 3, 6, 12, and
21. The obtained absorption capacity was proportional to
the cellular oxidoreductive activity and subsequently
scaled to 1 g hyaline cartilage. Measurement results
were evaluated using analysis of variance (ANOVA) and
Friedman’s and Dunnet’s statistical tests.
The research was approved by the Bioethical Com-
mission of the Medical University of Lublin (PL) no.
KE-0254/143/2006, according to Good Clinical Practice
None of the evaluated groups showed a statistically sig-
nificantly change in absorption from initial values during
the first 3 days of observation. Group IV (saline solution
enriched with glucosamine) had the highest absorption
values, both initially and during the subsequent evalua-
tion days, as calculated per gram of cartilage. A statisti-
cally significant decline in live chondrocyte numbers
occurred between Days 3 and 6 of storage in Ringer fluid
(group II) and saline with sodium salt of hyaluronic acid
(group III). A similar decline occurred in normal saline
(group I) between Days 12 and 21 of storage. No statis-
tically significant decrease in the number of live chon-
drocytes was evident in group IV (saline enriched with
glucosamine sulphate) over the entire observation pe-
At Day 21, group IV samples had the highest number
of live chondrocytes, and the difference in counts be-
tween this group and each other group was statistically
The lowest numbers of live chondrocytes throughout
the whole research period were found in group III, saline
solution with sodium salt of hyaluronic acid. The most
Table 1. Absorbance values of Ringer’s solution vs. other me-
Chondrocyte viability was highest in the 0.9% NaCl + glucosamine
group on all observation days.
Table 2. Trend analysis of absorbance values.
The lowest absorbance values and the biggest decrease in values were
seen in group III (0.9% NaCl + HAc). The highest absorbance values
and the smallest drop in values were seen in group IV (0.9% NaCl +
K. Gawęda et al. / HEALTH 2 (2010) 609-612
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
rapid drops in numbers of live chondral cells were also
observed in this group. The number of live chondral
cells in group II (Ringer solution) was lower than in
group I (normal saline solution).
The storage period of fresh osteochondral grafts greatly
affects their clinical value, where longer storage reduces
the number of live chondral cells [7].Allen et al. re-
ported a significant decline in live chondral cell numbers
in osteochondral grafts within 3 weeks of collection [4].
Previous research has focused on protecting chondrocyte
vitality in allogeneic osteochondral grafts primarily by
changing the temperature. Voss et al. demonstrated that
storing cartilage at room temperature accelerated cell
death [8], and a critical increase occurred between 50°C
and 55°C. In contrast, lowering the temperature is gen-
erally favorable to chondrocyte survival. Pearsall re-
ported that the number of live chondrocytes dropped to
67% of the initial value after a storage period of 44 days
at 4°C [9]. Judas et al. showed that adding protective
substances to the agents in which osteochondral grafts
were stored before freezing extended chondrocyte sur-
vival [10]. Adding tetracycline-type antibiotics may im-
prove the vitality of chondral graft cells [11]. Teng et al.
examined the impact of IGF-1(insulin-like growth factor
1) and the apoptosis inhibitor zVAD-fmk in Ringer fluid
and Dulbecco’s modified Eagle’s medium (DMEM) on
the survival of bovine chondrocytes [7]. Chondrocytes
atrophied most rapidly in Ringer fluid; the process was
slower in DMEM. Adding either IGF-1 or zVAD-fmk
to the DMEM significantly extended the survival of
chondral cells. Pennock analyzed the survivability of
human chondrocytes in osteochondral grafts suspended
in normal saline, glucose, amino acid solution, and 10%
fetal bovine serum solution [12]. Chondrocyte survival
was significantly lower in solutions without bovine se-
rum. However, storing tissue with bovine serum requires
further research on the possibility of the transmission of
infections as well as immune reactions to proteins from
foreign species.
The results of our research demonstrate that chondro-
cyte cell death was quickest in saline solution enriched
with sodium salt of hyaluronic acid. We are not in a po-
sition to determine why adding hyaluronic acid deriva-
tives did not improve chondrocyte graft vitality. Samples
of human joint cartilage stored for 3 weeks in saline so-
lution enriched with glucosamine sulphate retained their
vitality to the greatest degree compared to samples pre-
served in normal saline alone, Ringer solution, and nor-
mal saline solution plus sodium salt of hyaluronic acid.
Thus, chondrocyte vitality can be increased by enriching
the fluid environment of graft preservation with gluco-
samine sulphate. The lowest decline in live chondrocyte
numbers over a 3-week period decreased the biological
value of the graft only slightly. Our results show that the
inevitable process of in vitro chondral cell atrophy dur-
ing the storage period for surgical transplantation of joint
surfaces may be slowed to maintain high biological and
mechanical value of the graft. Otsuki et al. showed that a
decrease in glycosaminoglycan concentration did not
lead directly to the intensification of chondral joint cell
atrophy [13]. Based on our analyses, increasing the
amount may slow these processes.
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