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INTRODUCTION

The Temporomandibular joint (TMJ) connects the mandible to the skull and regulatesmandible movement. It is a bi-condylar joint in which the condyles fit in articularfossa. Between the condyle and the fossa-eminence of the temporal bone there is adense fibrocartilaginous disc that makes the TMJ an exceptional and unusual joint.

The TMJ disc is the major implicated on the joint function. The disc has a fundamentalrole during complex mechanical movements. In addition, the TMJ disc may serves tominimize shocks, distribute loads, and correct incongruence between the joints bonystructures.(1)The TMJ is one of the most complicated as well as most used joint in ahuman body, and therefore the TMJ pathology has an enormous impact in the life ofthe patients. (40)

The most common disorders are pain dysfunction syndrome, internal derangement,arthritis, and traumas, (2,3,4) and the treatment options for those disorders range fromphysical therapy and non surgical treatments to various surgical procedures: self-care, splints, arthrocentesis, arthroscopy, discectomy and finally joint replacementwith different implant devices. However, treatment options for TMJ patients havelimited success rates, especially with the synthetic TMJ disc implants, and it isnecessary to develop new methods of treatment.On this context, tissue engineering is a promising technology for the treatment of TMJdisorders allowing to get an engineered disc that serves to return the previouslydamaged joint to normal jaw function.(1)

The TMJ tissue engineering has an enormous potential, although this is an emergingfield still on develop. From the first study about TMJ disc cells in a tissue-engineeringenvironment, accomplished by Thomas et all. in 1991 (29), the fundamental issueshave been the selection of appropriate scaffolding material, cell sources, andbiological and biomechanical environments.(6,7,9,10,11,12,13,14,15)

As scaffolding materials, some studies had analyzed different biomaterials likehydrogel and porous scaffolds (8) or photopolymerizable and porous scaffolds (30,31,32)

The biological signals on TMJ disc constructs had been basically growth factor(platelet-derived growth factor, basic fibroblast growth factor, insulin like growthfactor (36), and elements like ascorbic acid (41)

Two known studies have utilized mechanical stimulation for the tissue engineering ofthe TMJ disc, Detamore and Athanasiou (42) used a rotating wall bioreactor, Almarzaand Athanasiou (43) observed the effects of hydrostatic pressure on TMJ disc cellsplated in monolayer.

Finally, the possible cell sources go from contralateral TMJ authologous cells, non-authologous cells, and different population cells with chondrogenyc potencial asfibroblastic cells (33) or stem cells (34,35) .

In addition to the previous statement about the cell sources, it is fundamental thatthese cells have a high viability. In this regard, to produce artificial organs and tissuesby tissue engineering is necessary an exhaustive evaluation of the viability ofcultured cells before they can be used, since only viable cells are suitable for clinicaluse. (Alaminos et all) This is important not only in human tissue engineering, but alsoin the construction of animal models (16).

Currently studies results have demonstrated that cell viability change notablybetween different subcultures. (alamino2007, morata, 2008) Some methods are focus on thedetection of cell membrane alterations, for example, by trypan blue or propidiumiodide staining, or by quantifying lactic dehydrogenase (LDH) in the culture medium(17,18).Nevertheless, most of these techniques are not accurate enough to detect earlycell damage, and they only identify cell irreversible alterations. In most cases a

positive result with these techniques reveals that the integrity of the cell membranehas been lost. For these reasons, such methods cannot detect cells that are prone to

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death but do not yet manifest cell membrane alterations. On In this framework,determining the cultured cells viability by quantification of the ionic content,especially potassium and sodium, is one of the most sensitive techniques (19,20,21,22).Certainly, the intracellular concentration of these ions correlates well with the vitalstatus of cells and is an excellent marker of cell physiology and cell viability. Electronprobe X-ray microanalysis associated with electron microscopy is the most powerful

approach to measure total elemental composition, making it possible tosimultaneously determine the concentrations of different elements and the ultrastructure of cells (23,24,25,26,27,28).

In this work, we evaluate the viability, of successive cell populations from differentchondrocytes subcultures, using the classical trypan blue method and the Electronprobe X-ray microanalysis associated with electron microscopy, in order to establishthe best population to be used in TMJ tissue engineering.

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MATERIALS AND METHODS:

Rat TMJ disc

The samples of TMJ fibrous cartilage were obtained from four adult Wistar white ratsweighting approximately 400 g. Later, TMJ was disectted, removing the jointligamentes to liberate the complete disc. Once we obteined the samples, these werequickly conserved in transport tisular medium at 4oC until the sample processing.This transport mdium was composed by DMEM (Dulbeccos modified Eaglesmedium, Sigma-Aldrich ref. D5796, St. Louis, Missouri, EEUU) supplemented withantibiotics and antimycotics solutions (G penicillin 500 U/ml, streptomycin 500 g/mland B anphotericin 1.25 g/ml, Sigma-Aldrich ref. A5955), without bovine serum.

This research was approved by the institutional experimentation committee, and all

animals were treated according to national and international guidelines on animalwelfare.

Isolation and culture of cartilage cells

To obtain separated cells from extracellular matrix, the samples were cut into smallpieces and treated with collagenase I of Clostridium hystoliticum (Gibco BRL LifeTechnologies Ref. 17100-017, Karlsruhe, Alemania) 2% at 37oC during 12 h. Then,chondrocytes were harvested by centrifugation at 100 rpm during ten minutes.

All cells were cultured in 25-cm2 Falcom tissue culture flasks with keratinocyteculture medium (QC). The QC culture medium was a mixture of DMEN (300 ml)(Sigma-Aldrich ref. D5796) and HAM-F12 (150 ml) (Sigma-Aldrich Ref. N6658)supplemented with cholera toxin (8 ng/ml) (Sigma-Aldrich Ref. C3012), adenine (24g/ml) (Sigma-Aldrich Ref. A9795), hydrocortisone (0,4 mg/ml) (Sigma-Aldrich Ref.H0888), insulin (5 mg/ml) (Sigma-Aldrich Ref. I2767), triiodothyronine (1,3 ng/ml)(Sigma-Aldrich Ref. T5516), fetal calf serum (50 ml) (Sigma-Aldrich ref. F9665),antibiotics and antimycotics solutions (G penicillin 100 U/ml de, streptomicin 100g/ml and B anphotericin 0,25 g/ml (Sigma-Aldrich Ref. A5955) (51). This medium hasdemonstrated being useful in the culture of different human and animal cells. (52,53,54)

The medium was changed once every 3 days.All the cell cultures were incubated at37oC with 5% Carbone dioxide. Subculture of the fibrocartilage cells was carried outwith trypsin 0.5 g/L-EDTA 0.2 g/L solution on subconfluent cell cultures. The cells werekept in culture up to the sixth subculture.

We decided study six passages because it has been previously suggested thatcultured cells begin to display morphological changes related to early senescencestarting from passages four to six. (44)

Viability Evaluation

Classic Trypan Blue method

To determine the cell viability by a coloring exclusion method, we employed thetrypan blue technique, counting the number of blue cells with a Neubauer counterchamber. All counts were done in triplicate, and the mean and standard derivations

were calculated for each subculture.

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Electron probe X-ray microanalysisFor X-ray microanalysis, subconfluent chondrocytes were subcultured using trypsin-EDTA on plated gold grids covered with a thin layer of Pioloform (polyvinyl butyral)(Ted Pella, Inc., Redding, CA) and sterilized overnight under UV light. Cells wereseeded at a density of 5,000 cells per grid and cultured in QN supplemented with10% serum, antibiotics and growth factors. After 24 h of culture on the gold grids

covered with Pioloform, support grids containing the endothelial cells were washed inice-cold distilled water for 5 sec to remove the extracellular medium. After washing,excess water was drained from the surface and the grids were immediately plunge-frozen in liquid nitrogen (46,47). After cryofixation, the grids were placed in a precooledaluminum specimen holder at liquid nitrogen temperature and freeze-dried atincreasing temperatures for 24 h in an E5300 Polaron freezer-drier apparatusequipped with a vacuum rotatory pump system. Freeze-dried gold grids were carbon-coated in a high-vacuum coating system and microanalyzed within 6 h.

Electron probe X-ray microanalysis of the specimens was performed with a PhilipsXL30 scanning electron microscope (SEM) equipped with an EDAX DX-4microanalytical system and a solid-state backscattered electron detector.

All determinations were performed on the central area of the cell nucleus. Todetermine total ion content, we used the peak-to-local-background (P/B) ratio method(48,49,50) with reference to standards composed of 20% dextran containing knownamounts of inorganic salts. (45)

In each chondrocyte subculture, we quantified 10 cells, from 3 different grids andthree different culture flaks

Statistical analysis:

To evaluate the statistical significance of the two consecutive subcultures, we usedthenonparametric MannWhitney test. Comparisons of several subcultures at a timewere carried out with the KruskalWallis test for multiples samples. For individualtests, a two-sided P-value less than 0.05 was considered statistically significant.

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RESULTS

Rat fibrocartilage cells culture

Isolated fibrocartilage cells proliferated rapidly in culture, and reached subconfluencyaround day 11 (11,5 2,8 days). In culture, fibrocartilage cells displayed a variablepolygonal, star and ever fusiform shape (Fig. 1)

Figure 1. Fibrocartilage cells used in this work. Left, phase contrast micrograph ofsubconfluent isolated fibrocartilage. Scale bar 200m. Right, confluents fibrocartilage cellsfixed with formaldehyde 4% and stained with hematoxylin y eosin. Scale bar 200m.

Cell viability using Trypan blue.

The obtained results are shown in the graphic 1 and in the table 1. These results

express the medium values, expressed in percentages, of viable cells again deadcells. In trypsinized cells, trypan blue staining demonstrated that most of the cells inthe first four subcultures were alive with viability above 90%, and that the percentageof live cells was slightly lower in the fifth and sixth subculture, 89.39 and 87.34%respectively.

When the values obtained by trypan blue method were compared among the sixsubcultures with the KruskalWallis test for multiple samples, the result wassignificant. One-to-one statistical comparisons of consecutive passages demonstratedthe different viability between the different sulcultures (table 2).

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Graphic 1. Evaluation with trypan blue method. Cell viability expressed in percentages of live

cells against dead cells.

SUBCULTURE

MEDIA ERROR STANDARD

1 99,17 0,45

2 95,06 2,36

3 97,37 2,56

4 98,61 1,04

5 89,39 1,16

6 87,34 0,65

Table 1. Medium values, expressed in percentages, of viable cells again dead cells and

standard deviation.

MANN-WHITNEY TEST KRUSKAL-WALLIS TEST

SUBCULTURE

S

1 vs 2 2 vs

3

3 vs

4

4 vs 5 5 vs 6 All 6 subcultures

TRYPAN

METHOD

P=0.0

37

N.S. N.S. P0.05.

Ionic content of cultured endothelial cells .

Analyses of 10 fibrocartilage cells from each subculture showed that culturedfibrocartilage cells had very variability K/Na ratios between different subcultures.Beginning with 4.58 for the first subculture, the K/Na ratio down in the second andthird subculture, 2.17 and 4.01 respectively, reaching the maximum value in the

fourth subculture with 4.65. k/Na ratio decreases in the final subcultures, 2.57 for thefifth and 1.17, the lowest value, for the sixth (graphic 2).

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Illustrative examples of spectra corresponding to cells from different subcultures areshown in Figure 3.

Graphic 2. Viability evaluation using Na/K ratios.

Table 2. Viability evaluation using Na/K ratios.

Table 3.All concentrations (in mmol/kg dry weight) are expressed as mean standard error

(SE).

K/Na

SUBCULTIVO

M

1 4,582 2,173 4,014 4,655 2,556 1,07

MEDIA STANDARD ERROR

SUBCULTURE

Na Mg P S Cl K Ca Na Mg P S Cl K Ca

1 137,16

92,59

934,55

199,89

318,61

628,17

20,81

78,39

47,12

513,71

107,93

178,47

363,44

9,12

2 246,

60

138,

60

1357,

19

401,

30

300,

90

536,

29

21,

49

133,

39

53,

52

359,

78

171,

13

86,3

0

179,

04

12,

153 179,

29119,72

1468,80

169,67

300,26

719,38

48,83

64,09

35,88

225,90

64,07

132,20

131,24

15,19

4 69,94

12,53

251,18

41,33

101,18

325,47

68,91

33,60

4,72

36,89

20,44

49,53

175,07

30,93

5 109,27

17,40

306,60

98,83

166,29

278,30

39,52

24,90

6,70

66,34

24,60

33,55

72,60

16,39

6 182,68

11,16

224,12

66,97

206,50

194,68

52,82

49,78

3,78

23,13

5,61 50,93

78,03

10,83

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Figure 3.Microanalytical spectra corresponding to a living fibrocartilage cultured cell. Thepeaks correspond to the energy dispersed by electrons located in the k orbitals of sodium(NaK), magnesium (MgK), phosphorus (PK), sulfur (SK), chlorine (ClK), potassium (KK), andcalcium (CaK).

When the micro analytical results were compared among the six subcultures with theKruskalWallis test for multiple samples, significant differences were found for all theelements analyzed, calcium, chlorine, magnesium, phosphorous, potassium, sodiumand sulfur ( P

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0.001 0.001 0.001 0.001

4 vs 5 P=

0.009

P=

0.004

N.S. P= 0.043 N.S. P=

0.007

P