Osteoarthritis and Cartilage (2000) 8, 279–287© 2000 OsteoArthritis Research Society International 1063–4584/00/040279+09 $35.00/0doi:10.1053/joca.1999.0302, available online at http://www.idealibrary.com on

Effects of chondroitin sulfate and interleukin-1� on human chondrocytecultures exposed to pressurization: a biochemical and morphologicalstudyF. Nerucci*, A. Fioravanti*, M. R. Cicero*, G. Collodel† and R. Marcolongo**Institute of Rheumatology, University of Siena, Siena, Italy†Institute of General Biology and Center for the Study of Germinal Cells, CNR, Siena, Italy

Summary

Objective: This study investigated the in vitro effects of chondroitin sulfate (CS) on human articular chondrocytes cultivated in the presenceor in the absence of interleukin-1� (IL-1�) during 10 days of culture with and without pressurization cycles.

Design: The effects of CS (10 and 100 �g/ml) with and without IL-1� were assessed in the culture medium of cells exposed to pressurizationcycles in the form of synusoidal waves (minimum pressure 1 Mpa, maximum pressure 5 Mpa) and a frequency of 0.25 Hz for 3 h byimmunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG). On the 4th and 10th day of culturethe cells were used for morphological analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

Results: The presence of IL-1� determines a significant decrease in PG concentration measured in the culture medium. When the cells arecultured in the presence of IL-1� and CS, a statistically significant restoration of PG levels is observed. Under pressurization conditions, weobserved that PG concentration in the medium of cells presents a significant increase at baseline conditions, in the presence of IL-1�+CS10and IL-1�+CS100, but not with IL-1� alone. The results concerning metabolic evaluation are confirmed by the morphologic findings obtainedby TEM and SEM.

Conclusions: These in vitro studies confirm the protective role of CS, which counteracts the IL-1� induced effects and they confirm theimportance of pressure on chondrocyte metabolism and morphology. © 2000 OsteoArthritis Research Society International

Key words: Osteoarthritis, Chondrocytes, Pressurization system, Chondroitin sulfate.

Received 12 March 1999; accepted 29 November 1999.This work was supported by IBSA (Institut Biochimique SA), Via

al Ponte 13 CH-6903 Lugano 3, Switzerland, who supplied thechondroitin sulfate.

Address correspondence to: Dr Fabiola Nerucci, Istituto diReumatologia dell’Universita di Siena Policlinico ‘Le Scotte’,I-53100 Siena, Italy. Tel: +39 577 233343; Fax: +39 577 40450.

Introduction

Osteoarthritis (OA) is one of the most common rheumaticdiseases directly involving the articular cartilage. The bio-mechanical properties of the cartilage depend upon theextracellular matrix produced by the chondrocytes, cellswhich are capable of maintaining a dynamic equilibriumbetween the anabolic and catabolic processes.1 The meta-bolic activity of these cells is regulated by several media-tors, such as cytokines, hormones and growth factors.2,3

Interleukin 1 (IL-1) is a cytokine involved in cartilagedegradation processes.4–8 It is produced by severaltypes of cells and can be found in the synovial fluid ofosteoarthritic subjects.9,10

The metabolic activity of chondrocytes is further influ-enced by mechanical factors depending on the articularload.11,12 At rest the hydrostatic pressure on the articularcartilage is about 2 atm; however, during ordinary physicalactivity it can increase to 100–200 atm.12

Several in vitro studies have shown the importance ofarticular load as a modulator of cartilage metabolism. In

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fact, the articular load influences the concentration ofproteoglycans (PG) and cations as well as the osmolarity ofthe cartilage microenvironment.13,14 These studies showthat chondrocytes respond to intermittent loading byincreased PG synthesis, whereas continuous mechanicalloading produces the opposite effect. The response isfurther modified by the magnitude, duration and frequencyof loading.15–17 Hydrostatic pressure affects proteins, thecytoskeleton and cell organelles; compression of cartilageresults in the deformation of cells and the extracellularmatrix, as well as physicochemical changes which includealtered matrix water content, fixed charge density, mobileion concentrations and osmotic pressure.11,13–17

The way in which loading is related to the incidence ofOA is not clear, although some studies have shown that lowlevels of intermittent fluid pressure, such as those thatoccur in vivo during Ilizarov joint distraction,18 have benefi-cial effects on joint tissue in OA, indicating that this factorcould be useful in the treatment of OA.19,20

Current OA treatment includes physical, pharmacologi-cal and surgical approaches. Pharmacological treatmentshave been divided into three categories: (1) analgesic andnonsteroidal anti-inflammatory drugs (NSAIDs); (2) symp-tomatic slow-acting drugs for OA; and (3) disease modify-ing drugs.21 The drug investigated in this study, sodiumchondroitin sulfate (CS), falls under the second category.21

CS is a glycosaminoglycan which is naturally found in theextracellular matrix of articular cartilage and is composed of

280 F. Nerucci et al.: Chondroitin sulfate on human chondrocytes

a long unbranched polysaccharide chain with a repeatingdisaccharide structure of N-acetylgalactosamine and glu-curonic acid.22 The benefits of the symptomatic effects ofCS in the treatment of OA have been tested in severalclinical trials.23–26

Chondrocyte cultures represent a valid and simplifiedbiological model in which to test the effects of drugs andsuch agents as cytokines, hormones and growth factors.They can be used also for the evaluation of the influence ofhydrostatic pressure on chondrocyte morphology andmetabolism.

In this work we have studied the in vitro effects of CS(Condrosulf®, IBSA Switzerland) on cultures of humanarticular chondrocytes cultivated in the presence or in theabsence of IL-1�, during 10 days of culture with and withoutpressurization cycles. Under these conditions we evaluatedthe effect of CS±IL-1� through metabolic (PG levels in theculture medium) and morphologic assessments carried outwith a transmission electron microscope (TEM) and ascanning electron microscope (SEM).

Materials and methods

The chondrocytes used in the study were grown inalginate gel following previously defined techniques.27,28

Alginate is a linear polysaccharide extracted from brownmarine seaweed, composed of 1–4 linked �-D-mannuronicacid and �-L-glucuronic acid.28,29 In our study we used a0.75% alginate solution that had been sterilized previouslyin an autoclave at 120°C for 20 min. In the presence ofbivalent cations, particularly Ca2+, alginate polymerizes toform a semi-solid support that can be used in the cultureand growth of chondrocytes. If necessary, the gelcan easily be dissolved and the cells can be used formorphologic and biochemical evaluations.

CHONDROCYTE EXTRACTION FROM HUMAN ARTICULAR

CARTILAGE

Human articular cartilage was obtained from the femoralheads of six OA subjects undergoing surgery for totalhip prostheses (mean age±SD of the patients: 67.5years±2.51, range 64–70 years). Immediately after sur-gery, macroscopically healthy cartilage were cut asepticallyand minced into 2-mm2 pieces. The cartilage fragmentswere washed in saline solution (A) (in mM: 140 NaCl,5 KCl, 5 glucose, 10 HEPES, pH 7.4) containing 200 U/mlof penicillin, 200 �g/ml of streptomycin and 0.25 �g/mlof amphotericin B. The cartilage was then digested byclostridial collagenase (Sigma, Italy) 1 mg/ml in PBS (inmM: NaCl 140, KCl 2.7, NaH2PO4 8.1, K2HPO4 1.5,pH 7.4) containing the same concentrations of antibiotics.The collagenase digestion was carried out at 37°C for14–18 h with moderate stirring. The solution was thenfiltered, washed in saline solution (A) and centrifuged for10 min at 700 g. As shown by the Trypan blue viable stain,90–95% of the recovered cells were alive.

Cells were then mixed with alginate (Pronova LVG,Protan, Drammer, Norway) in saline solution (A) to obtain afinal density of 5×105 cells/ml and 0.75% alginate. Aliquotsof 1 ml of alginate cell suspension were placed in 35-mmPetri dishes (Costar, Italy) containing a layer of previouslypolymerized alginate that prevented the cells from stickingto the bottom of the dish. Polymerization was obtained byadding 2 ml of CaCl 50 mM in 10 mM HEPES, 0.1 NaCl,

2

pH 7.4, which created a three-dimensional gel. Once thealginate had completely gelled, the excess calcium wasremoved and the gel containing the cells was washed twicewith saline solution (A).

Petri dishes were then overlaid with 2 ml of mediumcontaining 10% fetal calf serum (FCS), and 200 U/mlpenicillin, 200 �g/ml streptomycin, 2 mM glutamine and50 �g/ml ascorbate in Dulbecco’s minimum essentialmedium (DMEM) were added. The Petri dishes were main-tained in an atmosphere of 5% CO2 in air at 37°C and theculture medium was changed every 2 days.

STUDY WITH CHONDROITIN SULFATE

The effects of CS±IL-1� were analysed on chondrocytescultured as described above and the cultures were inocu-lated in Petri dishes (Ø35 mm) at a density of 5×105

cells/ml.The CS (25 KDa) was extracted from bovine cartilage.

Various analytical methods were used to demonstrate thehigh degree of purity of the product (>97%). CS wasextracted and purified by treatment with proteolyticenzymes and precipitation in the presence of organicsolvents. Electrophoretic separations of CS in agarose-gel30 and in Titan III31 did not detect contaminant gly-cosaminoglycans containing uronic acids. Less than 2%proteins or peptides were detected by Lowry assay.32 Lessthan 0.5 (w/w) nucleic acids were measured in thepreparation of the CS.

The CS (0, 10, 100 �g/ml), with or without human recom-binant IL-1� (Boehringer Mannheim, Italy) 5 ng/ml, wasadded to the culture medium throughout the test period.Every 2 days the medium was removed and stored at−70°C pending determination of its PG content.

PRESSURIZATION SYSTEM

Our pressurization system is different, in some aspects,from most of those currently in use.33 The pressure cham-ber is a hermetically sealed stainless steel cylinder with aheight of 400 mm and an internal diameter of 90 mm.Chondrocyte cultures in Petri dishes are filled with culturemedium, well sealed with a special membrane and thenplaced inside the pressure chamber. The chamber is filledwith distilled water and maintained by a thermostat at aconstant temperature of 37°C. The chamber is then press-urized by the oleodynamic energy produced by an electricrelay and applied, by means of a transfer accumulator, tothe water in the chamber. The operator can pre-set andchange the pressure inside the chamber during the testwith a data gathering and processing system installed in aPC and based on turbo Pascal language. The cells used inour tests were treated as described above. The Petri disheswere sealed with a Surlyn 1801 Bynel CXA 3048 bilayermembrane (thickness 90 �m; Du Pont, Italy) after excludingall air. The air in the dishes must be removed to avoidimplosions due to the presence of air which remainsbetween the membrane and the medium in the Petridishes. Surlyn membrane is partly permeable to O2 andCO2, but not to water or its other solutes. The membranewas attached to the rim of the Petri dish with Jet Melt 3764adhesive (3M, Italy). The chondrocytes covered in culturemedium were then pressurized according to sinusoidalwaves with a minimum pressure of 1 MPa, a maximumpressure of 5 MPa and a frequency of 0.25 Hz. The cells

Osteoarthritis and Cartilage Vol. 8 No. 4 281

were pressurized for 3 h every 2 days (days 2, 4, 6, 8 and10). After the pressurization phase, the medium was col-lected and stored at −70°C for PG determination. On the4th and the 10th days of culture some dishes were used formorphological analysis. After pressurization, the cells inalginate gel were immediately fixed for transmission elec-tron microscopy (TEM) and for scanning electronmicroscopy (SEM). Dishes that were not pressurizedserved as controls.

BIOCHEMICAL ASSAYS

The quantity of PG in the culture medium was measuredat different intervals (2, 4, 6, 8 and 10 days) by immuno-enzymatic method34 on microplates for the quantitativemeasurement of human PG (Medgenix Diagnostics,Belgium). This technique uses two monoclonal antibodieswhich are directed against the PG’s keratan sulfate, andanother monoclonal antibody which is directed against thebonding site of the PG’s hyaluronic acid. Standards andsamples containing PG react with the monoclonal antibody(MAbs1) attached to the well of the microplate, as well aswith another monoclonal antibody (MAbs2) labelled withhorseradish peroxidase (HRP). After an incubation periodwhich allowed for the formation of a sandwich made up ofcoated MAbs1/PG/MAbs2-HRP, the wells were washedseveral times in order to remove the labelled antibodywhich had not bonded. The amount of bonded antibodywas measured by adding and incubating a chromogensolution. The reaction was then blocked by adding a stopsolution. The microplate was read at 450 nm and thequantity of substratum was determined colorimetrically bymeasuring the absorbance, which is proportional to PGconcentration. The assay sensitivity was 0.9 ng/ml.

The DNA content in culture was analysed according toLabarca and Paigen’s35 fluorimetric method. This methodis based on the development of a fluorescent emissionwhen the fluorochrome-bis-benzimidazol reagent (Hoechstdye 33258) bonds with the DNA in culture.

TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

The cells were processed for morphological analysis onthe 4th and the 10th days of culture. For transmissionelectron microscopy, all samples were fixed for 2 h at 4°C incold Karnovsky fixative,36 rinsed overnight in 0.1 M pH 7.2cacodylate buffer and post-fixed for 1 h at 4°C in 1%buffered OsO4, dehydrated in a graded series of ethanoland embedded in Epon-Araldite. Ultrathin sections cut withan LKB III ultramicrotome were collected in copper grids,stained with uranyl acetate and lead citrate and thenphotographed with a Phillips CM10 electron microscope.We observed at least 50 cells from each group forevaluation.

For scanning electron microscopy, the cells in alginatewere fixed for 3 h at 4°C in Karnovsky fixative,36 washed incacodylate buffer 0.1 M at 4°C overnight, post-fixed in 1%OsO4 in veronal acetate buffer for 2 h and, after a briefwash in cacodilate buffer 0.1 M, placed in citrate pH 7.4 toremove the alginate. The chondrocytes were dehydrated ina graded series of ethanol, placed in tert-butanol andfrozen at 0°C before drying by sublimation of the tert-butanol in a vacuum chamber. The samples were sputtercoated with gold and observed in a Philips SEM 505. Weobserved at least 100 cells from each group for evaluation.

STATISTICAL ANALYSIS

The data were expressed as the mean±SD of PGrelease into the culture medium per micrograms of DNA inthe six tested cultures. The Student’s t-test was usedfor the statistical analysis: P<0.05 was consideredsignificant. When data were not normally distributed, theMann–Whitney U test was used.

Results

The total PG concentration in the culture medium duringthe 10 days of culture (pooled data from days 2, 4, 6, 8, 10)at baseline conditions, in the presence of a damagingstimulus represented by IL-1� at a concentration of 5 ng/mland in the presence of IL-1� and of CS at the twoconcentrations used in the study, i.e. 10 �g/ml and 100 �g/ml of culture medium, is shown in Fig. 1. The presence ofIL-1� determines a significant decrease (P<0.05) in PGlevels, but when the cells are cultured in the presence ofIL-1� and CS a statistically significant restoration (P<0.05)of PG concentration is observed.

In Fig. 2 we have compared the data shown in Fig. 1 withthose obtained under pressurized conditions. As Fig. 2shows, there is a significant increase (P<0.01) in the levelof PG in the culture medium in basal conditions. However,with pressurization there is a slight and insignificantincrease in the concentration of PG in the presence ofIL-1�. In pressurized conditions, the addition of CS inducesa significant increase (P<0.005 per CS10 �g/ml andP<0.003 per CS100 �g/ml) of PG in the culture medium.

The levels of PG in the culture medium at the differenttimes and in the different experimental conditions isdemonstrated in Table I. With the passage of time, therewas a progressive but not significant decrease in theconcentration of PG in the culture medium.

The morphological evaluations, obtained by TEM andSEM, seem to indicate an effect of IL-1 of the CS and of thepressure on the metabolic functions of the chondrocytes.Figure 3 shows chondrocytes photographed on the fourthday of culture by TEM. Figure 3A shows a cultured cell atbasal conditions: the nucleus appears euchromatic, thecytoplasm contains a fair amount of rough endoplasmicreticulum and lipid droplets, and the cytoplasmic processesof the plasma membrane extending out into the surround-ing matrix have abundant collagen fibres and PG granules.Figure 3B shows a cell cultured in the presence of IL-1�and its damage is evident: several vacuoles in the cyto-plasm are devoid of typical structures. The extracellularmatrix lacks both collagen fibers and PG granules. Figure3C shows a cell cultured in the presence of IL-1�+CS100 �g/ml and a clear restoration of the cell structurescan be observed: the nucleus shows a distinct nucleulus,the cytoplasm contains rough and smooth endoplasmicreticuli, a Golgi complex and lipid droplets. Figure 3Dshows a cell cultured at basal conditions and subjected topressurization: the abundant presence of collagen fibersand PG granules in the extracellular matrix are evident.

Figure 4 shows the SEM image of cells photographed onthe 4th day of culture. Figure 4A shows a cell cultured atbaseline conditions: it is interesting to observe that it hasretained its spherical shape, some secretion granules andthe thick network of collagen fibrils. Figure 4B shows a cellcultured in the presence of IL-1�: its damage is clearlyevident and this is further confirmed by the loss of cyto-plasmic processes and by the presence of superficial

282 F. Nerucci et al.: Chondroitin sulfate on human chondrocytes

Fig. 1. Total amount of PG measured in culture medium (ng/�g DNA) during 10 days of culture (pooled data) in basal conditions, in thepresence of IL-1� and in the presence of IL-1� and CS10 and 100 �g/ml. Data are expressed as mean values±SD b vs a; c vs b; d vs b:

P<0.05; *P<0.05.

Fig. 2. Total amount of PG measured in culture medium (ng/�g DNA) during 10 days of culture (pooled data) in basal conditions, in thepresence of IL-1� and in the presence of IL-1� and CS10 and 100 �g/ml with and without pressurization conditions. −pr=cyclicpressurization minimum pressure 1 Mpa and maximum pressure 5 Mpa at 0.25 Hz frequency. Data are expressed as mean values±SD e vs

a: *P<0.01; g vs c: **P<0.005; h vs d: ***P<0.003.

alterations. Figure 4(C) shows a cell cultured in the pres-ence of IL-1�+CS100 �g/ml: the cell morphology has beenpartially restored as confirmed by the presence of severalsurface granules. Figure 4D shows a cell cultured at basalconditions and subjected to pressurization: its SEM imageshows the presence of abundant matrix fibers and secre-tion granules. Figure 4E shows a cell cultured in thepresence of IL-1� and subjected to pressurization: itsdamage is shown in the absence of granules and the lackof a network of collagen fibers, although its characteristicspherical shape appears slightly restored. Figure 4F shows

a cell cultured in the presence of IL-1�+CS100 �g/ml andsubjected to pressurization: it shows the presence of abun-dant extracellular matrix fibers and the restoration of thecell surface morphology.

Figure 5 shows chondrocytes photographed on the 10thday of culture by TEM. Figure 5A shows a cultured cell atbaseline conditions. The ultrastructure is still as well con-served as it was on the 4th day of culture. Figure 5B showsa cell cultured in the presence of IL-1�+CS100 �g/ml andsubjected to pressurization: the cell shows a similar mor-phology on the 4th and the 10th days of culture.

Osteoarthritis and Cartilage Vol. 8 No. 4 283

Table IPG concentration (ng/µgDNA) in culture medium during the 10 day period in various experimental conditions

Days Basal Basal-pr IL-1 IL-1-pr IL-1+CS10 �g/ml

IL-1+CS10 �g/ml-pr

IL-1+CS100 �g/ml

IL1+CS100 �g/ml-pr

2nd 256.7±87.3 282.0±80.7 155.2±49.2 187.5±75.5 189.2±63.7 277.3±94.9 187.7±71.4 296.7±104.94th 215.5±26.7 245.3±55.6 152.0±41.9 178.5±50.4 199.3±43.7 239.0±60.6 187.3±52.1 250.3±46.1*6th 220.5±37.7 237.7±62.9 159.7±41.5 144.7±36.2 182.8±23.4 254.8±59.3*** 172.3±33.1 206.3±56.78th 169.8±65.9 236.5±64.2 137.5±44.6 152.2±36.9 174.7±49.5 224.2±88.8 148.5±37.5 194.8±78.9

10th 179.0±31.1 238.8±45.2** 155.0±42.6 146.8±50.6 188.0±50.2 202.7±87.6 184.5±55.6 197.8±78.1

*P<0.05 vs CS100 �g/ml; **P<0.02 vs basal; ***P<0.01 vs CS10 �g/ml.Data are expressed as mean values±SD.

Fig. 3. Sections of OA chondrocytes cultured in vitro for 4 days. (A) Basal conditions: the cell shows an euchromatic nucleus (N) and acytoplasm abundant in endoplasmic rough reticulum (RER). The plasma membrane (PM) has a large number of processes in a matrix richin collagen fibres and PG granules (arrows). Lipid droplets (LD); ×13,000. (B) Presence of IL-1�: the cell presents a very vacuolatecytoplasm (arrows). The matrix appears to be very poor. Nucleus (N), plasma membrane (PM); ×6000. (C) Presence of IL-1�+CS100: thecell clearly recovers its good state of health. The nucleus (N) presents a nucleulus (Nu). The cytoplasm contains rough and smoothendoplasmic reticuli (arrows); Golgi complex (GC) and lipid droplets (LD); ×14000. (D) Basal condition and pressurization: the abundantpresence of collagen fibres and PG granules (arrows) in the extracellular matrix is evident. Nucleus (N), lipid droplets (LD), plasma

membrane (PM); ×10000.

Discussion

In our study we have used alginate as the support in theculturing of human chondrocytes in considering the advan-

tages and draw-backs of most currently used tech-niques.37,38 Alginate gel is a valid alternative to otherculture techniques since it enables the chondrocyte toretain its 3D-structure and to maintain its characteristic cell

284 F. Nerucci et al.: Chondroitin sulfate on human chondrocytes

Fig. 4. Scanning electron micrographs of chondrocytes cultured in vitro for 4 days. (A) Basal conditions: the cell exibits a spherical shape andgranules of secretion are evident on its surface (arrows). The network of collagenic fibrils (CF) is also visible; ×3800. (B) Presence of IL-1�:the cellular damage is clear. The chondrocyte is devoid of granules and of matrix and the surface of the cell appears collapsed; ×4500. (C)Presence of IL-1�+CS100: the cell morphology has been partially restored. Newly formed elements of the extracellular matrix (EM) havebeen secreted into the extracellular space; ×5000. (D–F) Chondrocytes subjected to pressurization. (D) Basal conditions: the cell shows agood state of health; granules of secretion (arrows) and collagenic fibrils (CF) are evident; ×4500. (E) Presence of IL-1�: the cell is devoidof PG granules and of collagen fibres, but it does not appear collapsed; ×4500. (F) Presence of IL-1�+CS100: the cell shows a spherical

shape and a rich extracellular matrix (EM); ×3300.

shape, while preventing cell dedifferentiation.27,28 Thechondrocytes cultured with this technique are in a conditionwhich is the most similar to the in vivo situation and aretherefore valuable in drawing reliable morphological andmetabolic assessments.39–41

In this study we have tested the effects of IL-1� and CSin the presence and the absence of pressurization cycleson the morphology and metabolism of in vitro humanchondrocytes. These tests confirm the studies alreadycarried out on in vitro experimental models.39,42–44

It is currently accepted that IL-1 is an important mediatorin cartilage destruction. IL-1 is a prime up-regulator ofmetalloproteinase (MMP) gene expression by chondro-cytes, and it also down-regulates the production of theendogenous inhibitor of MMPs, the TIMPs as well as thesynthesis of PG and collagens.45

Our experiments have shown that the addition of IL-1determines a reduction of the concentration of the PG in theculture medium; this could be the result of the fact that

cytokine induces an inhibition of PG synthesis by thechondrocytes, as shown in the morphological analysis. Themorphological aspects show signs of cellular suffering withthe presence of vacuoles and the lack of cellular organellestypically responsible for the synthesis of the matrix glyco-proteins (the endoplasmic reticulum, the Golgi apparatusand mitochondria) (Figs 3B and 4B).

When the cells were cultivated in the presence ofIL-1�+CS there was a restoration of PG concentration inthe culture medium. This fact confirms the protective roleplayed by this substance which counteracts the IL-1�induced effects and might be used by the chondrocyte as asubstratum for the synthesis of PG.46

The articular cartilage is a tissue constantly subjected toa load which depends upon body weight and musculartension. The load also varies according to posture andphysical activity. Hydrostatic pressure is one of the severalfactors which operate in articular cartilage subjectedto loading.11,13–17,47,48 Hydrostatic pressure appears to

Osteoarthritis and Cartilage Vol. 8 No. 4 285

modulate aggrecan biosynthesis through membrane-mediated pathways, such as the transport of cations, aminoacids and macromolecules. It has also been suggestedthat hydrostatic pressure may alter the action of themembrane Na+/K+ pump, thus altering intracellular K+

concentrations.49

In our study, the pressure values applied were within thephysiological range of human joints. In fact, pressure peaksof up to 18 MPa have been measured in human hip joints;however, pressure levels of 5 MPa are most oftenencountered in the knee joint during normal gait.12,50

Chondrocytes undergoing physiological pressurizationaccording to sinusoidal waves with a minimum pressure of1 MPa, a maximum pressure of 5 MPa and a frequency of0.25 Hz presented a greater metabolic activity which wasexpressed in the increase of PG levels in the culturemedium at basal conditions (Fig. 2). This fact was alsoconfirmed in morphological analysis by TEM and SEM(Figs 3D and 4D). In our opinion the increase of PG in theculture medium, which was also observed in basal con-ditions, could be determined by a stimulation of cellular

activity induced by the pressurization. Relatively low hydro-static pressures have been shown to determine increasesin cyclic AMP and PG synthesis and decreases in DNAsynthesis.51 However, the signal transduction mechanismsthat lead to these metabolic changes must still be clarified.It is not clear that higher pressures, within the physiologicalrange, like those used in the present study, operate bythe same mechanism. The stimulating effect of the physio-logical hydrostatic pressure which we have shown is inagreement with that found by other authors who havealso utilized human osteoarthritic cartilage. Some studieshave shown that OA chondrocytes are more sensitive tophysiological hydrostatic pressure than normal chondro-cytes.19,20,52 The ‘stimulating’ effect of the pressure did not,however, manage to counterbalance the negative effectsdetermined by the addition of IL-1� which, in fact, induce aserious metabolic and morphological imbalance.

However, the addition of CS created a protection fromthe effects of IL-1� under the pressurization conditionsused in the study. The simultaneous use of pressurizationwith the presence of CS in fact determined a highlysignificant increase in PG concentration, as if a synergismof action exists between the drug and the mechanical factorregarding IL-1�. The biochemical data was also supportedby morphological analysis (Figs 4F and 5B).

The decrement in the release of PG in the culturemedium over time could be explained, in our opinion, by theentrapment of the PG in the cellular clusters. This asser-tion, in agreement with the findings of other authors,53,54

was confirmed in the morphological analysis by TEM on the10th day of culture. Figure 5 shows morphologically andultrastructurally well preserved cells that are organized in away that is similar to those observed on the 4th day ofculture.

In conclusion, the study confirms the importance ofpressure on chondrocyte metabolism. When subjected topressure, the cells behave differently towards both damag-ing and protective stimuli and this fact further supports thenecessity of studying these cells in vitro by recreating asituation that imitates as nearly as possible the in vivoconditions. Studies using chondrocyte cultures undergoingpressurization represent the most suitable experimentalmodels for understanding both the etiopathogeneticmechanisms of OA and which type of physical activity maybe best suited for the prevention of OA and for its therapy.

Our study points out the protective effects of CS on IL-1�in basal conditions and after pressurization of the chondro-cytes. These results confirm in vitro the data obtainedin clinical studies on patients with OA23–26 and onexperimental animal models of OA.55

Further studies are necessary in order to clarify themechanism of action of this drug and for the betterunderstanding of the importance of this approach in OAtherapy.

Fig. 5. Sections of OA chondrocytes cultured in vitro for 10 days.(A) Basal conditions: the cell shows an euchromatic nucleus (N)and a cytoplasm with abundant vesicles (V); ×13500. (B) Pres-ence of IL-1�+CS100 and subjected to pressurization: the cellshows a good state of health and the presence of PG granules(arrows) in the extracellular matrix is evident. Nucleus (N), lipid

droplets (LD), Plasma membrane (PM); ×10000.

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