Chondrocyte viability depends on the preservative solution

doi:10.4236/health.2010.26090

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Vol.2, No.6, 609-612 (2010) Health

doi:10.4236/health.2010.26090

Chondrocyte viability depends on the preservative

solution

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,

Poland

Received 11 February 2010; revised 15 March 2010; accepted 18 March 2010.

ABSTRACT

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

1. INTRODUCTION

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

610

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.

3. MATERIALS AND METHODS

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

conditions.

4. RESULTS

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-

riod.

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

significant.

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-

dia.

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 +

glucosamine).

K. Gawęda et al. / HEALTH 2 (2010) 609-612

611

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).

5. DISCUSSION

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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