ORIGINAL RESEARCH

Comparative Analyses of Pharmaceuticals or FoodSupplements Containing Chondroitin Sulfate: AreTheir Bioactivities Equivalent?

Antonietta Stellavato . Odile Francesca Restaino . Valentina Vassallo .

Rosario Finamore . Carlo Ruosi . Elisabetta Cassese . Mario De Rosa .

Chiara Schiraldi

Received: July 2, 2019 / Published online: September 7, 2019� The Author(s) 2019

ABSTRACT

Introduction: Oral supplementation of chon-droitin sulfate (CS) and glucosamine (GlcN),symptomatic slow-acting molecules, is recom-mended by European Society for Clinical andEconomic Aspects of Osteoporosis andOsteoarthritis and Musculoskeletal Diseases(ESCEO) and other European Union (EU)guidelines for the restoration of the articularcartilage surface in patients affected byosteoarthritis (OA). They are commercialized aspharmaceutical grade products and as foodsupplements in combination with plant extractshyaluronic acid, methylsulfonylmethane, and

other components. Food supplements do notneed to undergo the strict regulatory controls ofpharmaceutical grade products; thus, composi-tion and contaminants that could be presentmay not be evidenced before commercializationand these uncertainties may give rise to con-cerns about the bioactivity of theseformulations.Methods: In this paper 10 different food sup-plements (FS) from diverse European countrieswere analyzed in comparison with two phar-maceutical grade products (Ph) using updatedanalytical approaches and biochemical cell-based assays. The purity, the titer, and the ori-gin of CS in Ph and FS samples were initiallyassessed in order to successively compare thebiological function. Both food supplements andpharmaceutical formulations were testedin vitro, using the same final CS concentration,on primary chondrocytes and synoviocytes interms of (i) cell viability, (ii) activation of theNF-jB-mediated inflammation pathway, (iii)cartilage oligomeric matrix protein (COMP-2),IL-6, and IL-8 production.Results: All the FS presented a certain insolublefraction; the CS and the GlcN contents werelower than the declared ones in 9/10 and 8/10samples, respectively. All FS contained keratansulfate (KS) at up to 50% of the total gly-cosaminoglycan amount declared on the label.Primary cells treated with the samples diluted topresent the same CS concentration in themedium showed cytotoxicity in 7/10 FS while

Enhanced Digital Features To view enhanced digitalfeatures for this article go to https://doi.org/10.6084/m9.figshare.9448109.

A. Stellavato (&) � O. F. Restaino � V. Vassallo �R. Finamore � E. Cassese � C. Schiraldi (&)Department of Experimental Medicine, Section ofBiotechnology and Molecular Biology, University ofCampania ‘‘Luigi Vanvitelli’’, Via De Crecchio 7,80138 Naples, Italye-mail: antonietta.stellavato@unicampania.itC. Schiraldie-mail: chiara.schiraldi@unicampania.it

C. RuosiDepartment of Public Health, School of Medicineand Surgery ‘‘Federico II’’ of Naples, A.O.U. FedericoII of Naples, Via S. Pansini, 80131 Naples, Italy

M. De RosaBioteknet, Via De Crecchio 7, 80138 Naples, Italy

Adv Ther (2019) 36:3221–3237

https://doi.org/10.1007/s12325-019-01064-8

Ph preserved viability and reduced NF-jB,COMP-2, and secreted inflammatory cytokines.Conclusion: Among all samples tested, thepharmaceutical grade products demonstratedeffectivemodulation of biomarkers counteractingthe inflammation status and improving viabilityand the physiological condition of OA humanprimary chondrocyte and synoviocyte cells. Incontrast to that, most FS were cytotoxic at thetested concentrations, and only 3/10 of themshowed similarities toPh sample behavior in vitro.Funding: This work was partially supported byPON01_1226 NUTRAFAST, MIUR Ministerodell’Universita e della Ricerca Scientifica. Bio-teknet financed two short-term grants for grad-uate technicians. The journal’s Rapid Serviceand Open Access fees were funded by IBSA CH.

Keywords: Chondroitin sulfate; Foodsupplements; Glucosamine; HPAE-PAD;Human primary chondrocytes; Humanprimary synoviocytes; Keratan sulfate; NF-jBmediated inflammation pathway; OA in vitromodel; Rheumatology

INTRODUCTION

Osteoarthritis (OA) is essentially a debilitatingmusculoskeletal disease with a high public health

impact. Also known as degenerative joint disease,OA is characterized by a progressive loss of articularcartilage in synovial joints [1]. The major conse-quence is several functional limitations such asjoint stiffness, and it is associated with inflamma-tion of the synovial membrane. In fact, theimportance of synovitis in OA progression hasgradually been recognized [2]. It is estimated thatthis pathology affects 40million Europeans and 30million Americans over the age of 50 and it isexpected to increase byup to35%with the agingofthe population [3]. The pathogenesis of OA startswith a mechanical overloading of the articularjoints, cartilage degradation, joint space narrow-ing, and loss of cartilage and synovial fluids, andthus bone degradation [3, 4]. Specific mediatorsproduced during OA can directly act on cartilageand on multiple signaling pathways activatingother key inflammatory molecules [4]. In fact, thenuclear factor kappa-light-chain-enhancer of acti-vated B cell (NF-jB) pathway is associated with OApathophysiology [4, 5], prompting the expressionof the inflammatory mediators (Fig. 1). Theseinclude interleukin-1 beta (IL-1b), tumor necrosisfactor alpha (TNF-a), interleukin-6 (IL-6), inter-leukin-8 (IL-8), cyclooxygenase-2 (COX2),chemokines, and colony-stimulating factors [6, 7].

Current OA pharmacological therapy isdirected at the prevention of pain and theimprovement of function. Standard treatments

Fig. 1 Schematic view of the experimental setup

3222 Adv Ther (2019) 36:3221–3237

mainly include a combination of pharmacolog-ical and non-pharmacological methods, likephysical exercises, physiotherapy, weight loss,etc. The first choice has been pharmacologicaltherapy with analgesics or non-steroidal anti-inflammatory drugs (NSAIDs) essentially direc-ted towards the prevention and the control ofpain and the improvement of motility functionin patients affected by OA [8]. However, numer-ous studies reported that the prolonged use ofNSAIDs could cause adverse effects in the gas-trointestinal and cardiovascular systems. For thisreason—and bearing in mind that the patienttreatment is prolonged for years—the need hasdeveloped for new therapeutic targets for OAtreatment that could relieve pain and potentiallyreduce the damage. As an alternative to NSAIDs,chondroitin sulfate (CS) and glucosamine(GlcN),which are natural extractive compounds,have been adopted [9, 10] since the 1990s and

recently recommended in the European Societyfor Clinical and Economic Aspects of Osteo-porosis and Osteoarthritis and MusculoskeletalDiseases (ESCEO) algorithm as preferentialtreatments at the initial stage of OA. Severalclinical trials have reported the efficacy of CS andGlcN in delaying joint damage, in pain reduc-tion, and in improving articular mobility inpatients with OA. Unlike NSAIDs, prescriptionCS and/or GlcN drugs are able to delay the pro-gression of the pathology or eventually reversemorphological changes in joint structure besidesworking as Symptomatic Slow-Acting Drugs forOA (SySADOAs) and Disease-ModifyingOsteoArthritis Drugs (DMOADs) [11]. The rec-ommended daily dosages for CS are between 800and 1200 mg while the daily dosages for GlcNrange from1250 to 1500 mg (Table 1). At presentthey are recommended by the European LeagueAgainst Rheumatism (EULAR) and ESCEO as

Table 1 Chondroitin sulfate and glucosamine origin, structure, and suggested posology

Chondroitin sulfate (CS) Glucosamine (GlcN)

Structure

OH

H

OR

OO

ROO

H

HO

H

HOH

HO

H

H

HNHAcH

HCOOH

O

R=SO3-

n

4)-b-GlcA-(1?3)-b-GalNAc-(1?

Terrestrial origin CS Mw = 14–26 kDa; 4S/

6S = 1.3–5.0

Marine origin CS Mw = 30–80 kDa; 4S/

6S = 0.3–1.8

[17, 18]

O

H

H

HOHH

OH

OH

H

HO

H

HO

GlcN Mw = 215 Da

Biological role The CS is a glycosaminoglycan (GAG) found in the

extracellular matrix of the animal cartilaginous

tissues

In the human body the GlcN is produced as

precursor of glycosylated lipids and proteins,

GAGs, and proteoglycans

Origin CS is manufactured by extraction and purification

from the animal cartilaginous tissues of shark fins,

bovine and chicken trachea, or pig muzzle

GlcN is manufactured from chitin of crustacean

shells by both alkaline or acid hydrolysis or by de

novo chemical synthesis, and it is generally sold as

glucosamine hydrochloride or glucosamine sulfate

salts [9]

Recommended

daily dosage

800–1200 mg 1250–1500 mg

Adv Ther (2019) 36:3221–3237 3223

SySADOAs for the treatment of knee, hand, orhip osteoarthritis, providing pain relief andincreasing joint mobility [12, 13]. Numerousfood supplements containing CS and/or GlcNare on the market and they are becoming anincreasingly popular alternative approach toprescription drugs especially among thosepatients who prefer non-pharmacological treat-ment options. However, there is still an unsolvedproblem related to the quantitative and qualita-tive control of CS and GlcN contained in foodsupplements and nutraceuticals [14–16].

In the absence of any mandatory analyticalcontrols from European Medicines Agency (Eur.Pharmacopeia) and of other specific national/international regulatory rules or of specificindustrial guidelines, each manufacturer hasbeen able to establish its own analytical proce-dures for the determination of chondroitinsulfate and glucosamine identity, content, andpurity in food supplements [19–22]. Recentpapers have also demonstrated that keratansulfate (KS), which is another component ofanimal articular cartilaginous tissue, may beextracted along with the CS, thus contaminat-ing it [23]. Because of the structural similarities,and the superimposable size of CS and KSbiopolymers, the selective removal of thismacromolecule is challenging [24–26]. It is ofgreat scientific interest for the assessment ofanalytical strategies to fully characterize foodsupplement purity, with the specific target offinding a correlation between structural char-acteristic and biological activity using in vitromodels [27]. In fact, robust data, generated inwell-assessed models, may better permit thecritical analysis of the clinical scientific litera-ture to ascertain the validity of the guidelineson a strong scientific basis. Often the SySADOAshave been regarded as ‘‘questionable’’ productswith controversial clinical outcomes. However,it was pointed out that the variability in thebeneficial effects in patients with OA is stronglyreduced, ascertaining effectiveness of CS-basedtreatments, when referring to pharmaceuticalgrade products [28].

In this study, 10 FS commercialized in eightdifferent European countries were extensivelyanalyzed and compared to pharmaceutical CS

with a specifically assessed experimental designon three human primary cell models.

METHODS

This article is not based on in vivo studies, oneither animals or humans. However, the cellsused were obtained by digestion of joint/carti-lage tissues harvested from surgical procedureson human participants. The pieces would havebeen wasted and, as part of a collaborationscheme, these were used instead to set up in vitromodels used in this experimental research pro-ject. The protocol was approved by the ethicalcommittee of the University (AOU-SUN) (proto-col registration No. 0003711/2015 of the 16/05/2015; study title ‘‘Glicosamminoglicani e/oderivati biotecnologici utili alla rigenerazionetessutale’’). Informed consent was obtained fromall participants involved in this study.

Reagents

Sodium nitrate, sodium acetate, N-acetylgalac-tosamine, glucosamine, galactose, and glu-curonic acid were from Sigma–Aldrich (USA).The NaOH solution was from J.T. Baker(Netherlands). The hydrochloric acid was fromCarlo Erba (Italy). The medium and theenzymes used in the biological experiments forcell cultures were from Gibco, Invitrogen (USA),unless otherwise specified. The 10 food supple-ments (FS), containing both CS and GlcN, andthe two pharmaceuticals (Ph), containing onlyCS, that were from different European countriesand companies, were either purchased orobtained as test/gift samples. Cell culturereagents and enzymes used in the biologicalexperiments were from Gibco, Invitrogen(USA), unless otherwise specified.

Analytical Evaluation: StructuralCharacterization and Composition

A single dose of FS (one capsule or tablet) wasdissolved in 20 ml of MilliQ water, in triplicate,placed and stirred at 300 rpm, at room temper-ature for 6 h and then left overnight to

3224 Adv Ther (2019) 36:3221–3237

sediment. The next day, the samples werephotographed and then centrifuged at6500 rpm for 30 min (Avanti J20-XP, BeckmanCoulter, USA) to separate the supernatants fromthe insoluble fractions which were then driedunder vacuum at 40 �C for 24 h (Binder GmbH,Germany) and weighed. The insoluble solidweight values were calculated as a ratio of theweight of the insoluble portion of the sample tothe total dry weight, as a percentage. Thesupernatants were filtered on 0.22-lm filters(Millipore, France) and the protein content wasdetermined according to the Bradford method[29], following a protocol previously described[23].

Chromatographic Profiles by Strong AnionExchange (SAX) Chromatography

Each sample (one capsule or tablet) was dis-solved in 20 ml of buffer A (20 mM sodiumacetate, 0.5 M sodium chloride, pH 7.4), cen-trifuged at 6500 rpm for 30 min (Avanti J20-XP,Beckman Coulter, USA), and the supernatantwas separated from the insoluble portion. Foreach sample, 2 ml of the supernatant weremicrofiltered on 0.22-lm filters (Millipore,France) and loaded on an anion-exchange col-umn (HiPrep Q Sepharose 16/10 HP, GEHealthcare, Milan, Italy) connected to an AKTApurifier system (GE Healthcare, Milan, Italy),previously equilibrated with buffer A. Sampleswere then eluted with buffer B (20 mM sodiumacetate, 3.0 M sodium chloride, pH 7.4) apply-ing a linear gradient. The chromatograms wereobtained by recording a signal at 215 nm andthe peak areas were used to calculate the rep-resentativeness of each peak in each sample asthe percentage ratio of the area of the singlepeak divided by the sum of the areas of all thepeaks present in the chromatograms [23]. Piecharts were created by using these instances ofrepresentativeness.

HPAE-PAD Analyses

Determination of the CS and GlcN contents ofthe sample supernatants and of their eventualKS contamination was performed by high-

performance anion-exchange chromatography(HPAE-PAD) analysis on the basis of themonosaccharide composition, according to apreviously reported method [23]. The CS con-tent was determined after a hydrolytic proce-dure on the supernatants as previouslydescribed [23]; the GlcN monosaccharide con-tent was determined after ultrafiltering thesample supernatants on 3-kDa membranes,according to a previously reported protocol[23, 26] and analyzing the permeate fractions.The retentate fraction that contained the CSand the eventual KS was hydrolyzed and ana-lyzed as well. Quantitative analyses were basedon determination of the GalNAc concentrationfor the CS content, of the free GlcN concentra-tion for the GlcN monosaccharide content, orof the bound GlcN concentration for the KScontamination.

Biological Activity

Bioactivity of food supplements in comparisonto CS pharmaceutical-based products wasassayed using three different cellular models:(I) chondrocytes isolated from nasoseptal carti-lage of healthy human patients and exposed toIL-1b (10 ng/mL) in order to mimic in vitroinflammatory conditions [30, 31]; (II) articularchondrocytes; and (III) synoviocytes that wereisolated from knee cartilage and synovial fluidof patients affected by OA who underwent ajoint replacement surgical procedure at theOrthopedics and Traumatology Department ofthe Federico II University of Naples, as previ-ously reported [32].

Isolation and Culture of CartilageChondrocytes and Knee JointSynoviocytes

1st ModelNasoseptal cartilage was obtained from surgicalprocedures on functional respiratory airways.Patients did not present cartilage alteration(e.g., OA, etc.). The protocol assessed in our labwas previously reported [30]. Briefly, mincedcartilage was digested using an enzymatic solu-tion (collagenase type I at 3 mg/ml and dispase

Adv Ther (2019) 36:3221–3237 3225

at 4 mg/ml), diluted in PBS, and gentamicin0.2 mg/ml (Hospira, IL, USA) at a temperature of37 �C on a shaking plate overnight. The cellswere separated from undigested pieces througha sterile filter (70 lm, BD Biosciences Bedford,USA) and the cellular suspension was cen-trifuged at 1500 rpm for 7 min (EppendorfCentrifuge, Milan, Italy). Cells were thenwashed with PBS, re-centrifuged, and re-sus-pended in DMEM supplemented with fetalbovine serum (FBS, 10%). Fluorescence-acti-vated cell sorting (FACS) analyses were per-formed to characterize the cell phenotype [30].

2nd and 3rd ModelsIn particular, two different kinds of cartilage fromthe same knee were obtained: a piece of intactcartilaginous tissue that represents the healthypart and was considered the control (healthycontrol), while the other samples were harvestedfrom damaged cartilage tissue of the same knee(pathological control). Serum (FBS) (10% v/v),penicillin–streptomycin (1% v/v), and ampho-tericin B (1% v/v) (Lonza, Basel, Switzerland).

Digestion was accomplished following thesame protocol describe above, and previouslypublished [32]. The cells were seeded in a35-mm tissue culture well and maintained at37 �C in a humidified atmosphere with 5% v/vCO2. Synoviocyte cells were isolated from thesynovial fluid. Specifically, synovial fluid sam-ples were centrifuged, the supernatant wasremoved, and the pellet was washed with PBS.The derived cells were seeded, after characteri-zation using flow cytometry according to thepreviously reported protocol [32, 33].

Cell Viability Assay

All three models described above were used for aviability assay in the presence of the FS and Phproducts. Trypan blue exclusion assay was usedto discriminate viable cells from non-viableones; 2 9 104 cells were seeded in 12-well plates(BD Falcon, USA) to obtain a consistent mono-layer. In particular, after 48 h of incubation theculture medium was removed and then replacedby either fresh medium alone (control), ormedium containing the food supplements or

the pharmaceutical grade chondroitin sulfatesamples. All the samples (FS1, FS2, FS3, FS4, FS5,FS6, FS7, FS8, FS9, FS10, Ph1, and Ph2) weretested at the same CS concentration (4 mg/mL).After 48 h, cells were harvested with trypsin,centrifuged, and stained with trypan blue(Sigma Aldrich, Milan, Italy) following themanufacturer’s instructions. Viable cells (notcolored) and dead cells (blue) were counted byusing a Burker chamber from two differentindependent observers. Cell viability (%) wasexpressed as the number of viable cells/thenumber of total cells 9 100.

Quantification of IL-6 and IL-8 ProductionUsing ELISA

Cell supernatants were collected after 48 h oftreatment, centrifuged (3000 rpm for 10 min at4 �C), and analyzed to quantify IL-6 and IL-8production. The cytokines were assayed usingan ELISA (Boster Biological TechnologyPleasanton, USA). Each experiment was per-formed in triplicate and cytokine amounts weredetermined using a microplate reader (Bioradlaboratories, Milan, Italy). The analytic con-centrations were calculated using a standardcurve according to the manufacturer’s instruc-tions and as previously reported [33].

Protein Extraction and Western BlotAnalyses

The cells’ intracellular total protein content wasextracted in the radioimmunoprecipitationassay (RIPA buffer) (19) (Cell Signaling Tech-nology, USA). Protein concentration was deter-mined using the Bradford method (BioradLaboratories, Milan, Italy) and 5 lg of proteinswas separated by means of sodium dodecylsulfate polyacrylamide gel electrophoresis,transferred onto nitrocellulose membranes(Millipore, Bedford, MA), and blocked with 5%skimmed milk in Tris-buffered saline withTween 20 (TBST). The membranes were incu-bated with primary antibodies to detect COMP-2 (Abcam, Cambridge, UK) and NF-jB (SantaCruz Biotechnology, CA, USA) both used at1:500 dilutions and incubated overnight at 4 �C.

3226 Adv Ther (2019) 36:3221–3237

After washing with TBST, immunolabelingbands were detected by the chemiluminescencedetection system using corresponding horse-radish peroxidase-conjugated secondary anti-bodies (Santa Cruz Biotechnology, CAUSA),diluted 1:10,000 for 1 h at room temperatureand reacted with an ECL system (Merck Milli-pore, Darmstadt Germany). Protein levels werenormalized with respect to the signal obtainedwith an anti-actin antibody 1:500 dilution(Santa Cruz Biotechnology, CA, USA). The semi-quantitative analyses of protein levels werecarried out using the Gel Doc 2000 UV System(Biorad laboratories, Milan, Italy) according tothe manufacturer’s protocols and previouslyreported [32, 33].

RESULTS

Insoluble Solid Determination and ProteinContent Analyses

As can be seen from Table 2, Ph and FS sampleslook very different in terms of color, trans-parency, and sediment. In fact, FS solubility islower than that of analyzed Ph products. Thelatter showed transparent rather than coloredsolutions while FS suspensions were yellow-or-ange colored and opaque (photo in Table 2).The 10 FS samples were analyzed to evaluate theprotein content. In sample FS1 the quantity ofproteins found is 3.9% of the declared value.The presence of proteins was identified in threeof the five FS samples (Table 2) even if notclearly stated on the labels.

Chromatographic Profiles by SAXChromatography

The chromatographic profiles of the FS samplesobtained by SAX chromatography were used tocreate a pie chart in which the percentage ratioof the area of every single peak divided by thesum of all the peak areas was reported (Fig. 2).Generally three families of peaks were visible inthe chromatograms: the unretained (UR) peakcontaining the species not bound to the sta-tionary phase, the chondroitin sulfate (CS) peak,

and the other component peaks (OC). A repre-sentative chromatogram is shown in Fig. 2. Asvisible in the graphs (Fig. 2), in Ph1 and Ph2samples the CS peak had a percentage areahigher than 80% while among the FS samplesonly FS5 showed a CS peak higher than 60%.The others showed lower CS percentages withthe lowest value of 3.1% being in the case of FS7.

CS, GlcN Content, and KS Contaminationby HPAE-PAD Analyses

The CS and GlcN contents in the food supple-ments and the eventual residual KS contami-nation were determined by HPAE-PAD analysesand data were compared with the pharmaceu-tical grade samples. The CS content in the foodsupplements was lower than the declared con-tent in nine samples out of 10, with differencesranging from 5.0 to 145.6 mg (Table 2); theGlcN content was lower the declared contentsin eight samples out of 10 with differencesranging from 3.0 to 130.4 mg (Table 2). All thesamples were contaminated with KS in therange from 11.7% to 47.9% of the total amountof GAGs present (Table 2). The pharmaceuticalgrade samples showed a CS content consistentwith the declared content and a KS contami-nation of 2.0% maximum (Table 2).

Biological Activity

First Model: Nasal ChondrocytesCell Viability Food supplements were assayedon IL-1b-challenged nasal chondrocytes[29, 32]. Cell viability was slightly reduced inthe presence of IL-1b (65%) with respect tocontrol. Ph1 and Ph2 particularly improvedviability and among the FS tested only three(FS2, FS3, and FS6) supported growth. Specifi-cally, the percentage of viable cells found for allthree was in the range 60–70%, with respect tountreated cells (CTR) (Fig. 3a).

Cytokine Quantification: IL-6 and IL-8 ProteinExpression in Cell Supernatants Resultsshowed that, as expected, IL-1b treatmentincreased both IL-6 and IL-8 level by about 2.5-fold and 1.6-fold, respectively, with respect to

Adv Ther (2019) 36:3221–3237 3227

Table2

Insolubledryweight,declared

andquantifiedchondroitinsulfateandglucosam

inecontents,keratan

sulfatecontam

ination,plantextractpresence,declared

andquantifiedproteincontentof

the10

Europeanchondroitinsulfate

food

supplementsandof

thetwopharmaceuticalproducts

Sample

Insoluble

dryweight

(%)

DeclaredCS

content

(mg/do

se)

Quantified

CS

content

(mg/do

se)

Declared

GlcN

content

(mg/do

se)

Quantified

GlcN

content

(mg/do

se)

KS

contam

ination

[%KS/

(CS1

KS)]

Plant

extracts

Declared

protein

content

Protein

amou

nt/

proteindeclared

(mg/mg)

Ph1

0400.0

391.9±

2.0

––

2.0±

0.1

No

No

Nd

Ph2

0400.0

394.0±

1.6

––

1.5±

0.1

No

No

Nd

FS1

2650.0

55.8

±1.6

750.0

619.6±

2.6

36.5

±0.3

Boswellia

s.,Salix

a.,

curcum

in

Yes

Nd/0.02

FS2

32166.0

161.0±

7.8

410.0

340.2±

1.5

23.1

±0.2

No

No

Nd

FS3

39365.0

296.8±

4.2

416.0

453.2±

0.5

16.8

±0.2

No

No

0.27

FS4

5300.0

163.8±

0.9

375.0

396.2±

1.0

47.9

±2.8

Harpagofitum

No

0.57

FS5

4300.0

212.8±

5.2

375.0

372.0±

1.2

11.7

±0.7

Curcuma

Yes

Nd/10

FS6

33400.0

254.4±

0.8

290.0

216.1±

1.6

15.5

±0.1

Boswellia

s.No

0.72

FS7

31250.0

200.2±

8.9

400.0

374.6±

0.7

14.1

±0.5

No

Yes

Nd/60

FS8

4400.0

260.6±

0.6

500.0

446.9±

1.4

40.6

±0.4

No

Yes

Nd/180

FS9

35150.0

100.8±

4.1

500.0

417.8±

2.8

33.7

±0.5

No

Yes

0.39/10

FS10

38200.0

160.0±

0.7

600.0

527.6±

1.0

23.3

±0.7

No

No

Nd

Ndnotdetected-und

erdetectionlim

itPictureof

thepharmaceuticalproductsandfood

supplementinsolublecontents

Ph1

P

h2

F

S1

FS

2

FS3

FS4

FS

5

FS6

F

S7

FS8

F

S9

FS1

0

3228 Adv Ther (2019) 36:3221–3237

CTR. Beside, Ph1 and Ph2 significantly reducedIL-6 expression by 5-fold and 4.5-fold, respec-tively, with respect to IL-1b challenged cells.Among FS samples only the three that provednot to be cytotoxic at the concentrations testedshowed their efficacy as anti-inflammatoryagents. In fact, FS2, FS3, and FS6 reduced IL-6production with respect to IL-1b treatments(Fig. 3b). For IL-8, both pharmaceutical prod-ucts, Ph1 and Ph2, reduced production by 2.4-fold and 9.8-fold, respectively. Among foodsupplement samples, FS6 was not able to reduceIL-8 expression, whereas FS2 and FS3 reducedIL-8 by about fivefold with respect to the nega-tive control (Fig. 3b).

Second Model: Articular chondrocytesCell Viability Cell viability was lower for pri-mary chondrocytes (Fig. 4a) isolated from verydamaged joint cartilage of patients with OA(pCTR) with respect to the ones isolated fromless damaged parts (considered as healthy). Ph1and Ph2 improved the condition, with 65–75%of counted viable cells. Among the FS tested,only three (FS2, FS3, and FS6) proved not

detrimental to cell growth, providing at least65% of viable cells as reported in Fig. 4b.

Cytokine Quantification: IL-6 and IL-8 ProteinExpression in Cell Supernatants Results ofELISAs (Fig. 4c) showed that IL-6 of pCTR wasincreased with respect to the healthy one asexpected. Ph1 and Ph2 both reduced this mar-ker by about twofold. Among the three FSselected, FS2 does not show any effect but FS3prompted a 1.7-fold reduction with respect topCTR while FS6 reduces IL-6 by 1.2-fold. Con-cerning IL-8 expression, data showed that onlyPh1 and Ph2 reduced this marker level withrespect to pCTR by about 2.5-fold and 1.5-fold,respectively. In contrast, the food supplementsshowed a negative modulation of thisbiomarker.

COMP-2 and NF-jB Protein Expression: Wes-tern Blotting Experiments The cells obtainedfrom damaged cartilage (pCTR) expressedhigher levels of the pro-inflammatory proteinNF-jB and COMP-2 than hCTRL, as evidencedby western blotting analyses (Fig. 4d). After 48 h

Fig. 2 a FS4 strong anion exchange chromatographyprofiles: in blue, absorbance at 215 nm; in red, at280 nm. b Graphs obtained from the ratios of the peakareas at 215 nm: UR unretained peak, OC other compo-nent peak, CS chondroitin sulfate peak. UR peak may

contain non-sulfated CS, KS, and/or other components;OC peaks may contain glucosamine, proteoglycans, andkeratin sulfate (in the last peak); CS peak containschondroitin sulfate as reported by [23]

Adv Ther (2019) 36:3221–3237 3229

of treatment both with pharmaceutical prod-ucts (Ph1 and Ph2) and food supplements, NF-jB and COMP-2 protein expression decreased.In particular, Ph1 and Ph2 showed a 1.3-foldand 1.5-fold reduction of NF-jB expression incomparison to pCTR, respectively. However,among the three FS that were not cytotoxic, FS3was also able to reduce NF-jB expression byabout 1.6-fold. Furthermore, as shown inFig. 4d, Ph1 and Ph2 also slightly reduced theexpression of COMP-2 by about 1.3-fold and1.2-fold, respectively, with respect to pCTR.Among FS, FS6 reduced COMP-2 expression by1.5-fold with respect to p-CTR.

Third Model: SynoviocytesCell Viability The synoviocyte cell (Fig. 5a)based model does not have a healthy control

since the cells are isolated from a population thatis consistent with the pathological OA condition(membrane and fluid in the OA-affected joint).Cell viability (Fig. 5b) is about 65 ± 7%after 48 hfor the pCTR, as expected. Ph1 and Ph2 treat-ments improved viability by up to 85%. AmongFS tested, the cell viability percentage for FS5 isabout 20%, while for FS6 it is about 65%. Resultsfor FS2 and FS3 were better, with percentage ofviable cells of about 80 ± 10% for both.

Cytokine Quantification: IL-6 and IL-8 ProteinExpression in Cell Supernatants In synovio-cyte cells, IL-6 production (Fig. 5c) is signifi-cantly reduced in the presence of Ph2 by about35-fold with respect to pCTR. However, Ph1 isalso able to counteract cytokine production,specifically by 2.2-fold with respect to pCTR.Concerning food supplements, only the sam-ples that reduce cell viability were consideredfor IL-6 quantification. In particular, FS2reduced IL-6 production by about 1.6-fold withrespect to pCTR; FS6 reduced IL-6 production by2.2-fold, and FS3 reduced IL-6 production byabout 30-fold.

Concerning IL-8, Ph2 proved the mosteffective but all the FS-treated samples affordedIL-8 concentrations not significantly modulatedwith respect to pathological control.

COMP-2 and NF-jb Protein Expression: Wes-tern Blotting Experiments We evaluated themodulation of the same biomarkers for the thirdimplemented model. In this context theobtained cells were regarded as being subject toan ongoing inflammatory process. After 48 h of

Fig. 3 a Nasal chondrocyte cell viability using trypan bluestaining. b IL-6 and IL-8 cytokine quantification usingELISA. T test analyses (*p\ 0.01) were performed tocompare the significance of both pharmaceutical gradechondroitin sulfate samples and food supplements on IL-6and IL-8 production by nasal chondrocytes with respect toIL-1b-treated cells

cFig. 4 Biological activity on articular chondrocytes. a Cellpicture panel in the presence of food supplements andpharmaceutical grade products versus healthy and patho-logical CTR. b Cell viability using trypan blue staining.c IL-6 and IL-8 cytokine quantification through ELISA.d Western blotting analyses of NF-jB and COMP-2versus actin housekeeping protein normalization. T testanalyses (*p\ 0.01) were performed to compare thesignificance of both pharmaceutical grade CS samplesand food supplements on IL-6 and IL-8 production, onNF-jB and COMP-2 protein expression by articularchondrocytes with respect to pathological non-treated cells

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treatment, Ph2 was more effective than Ph1with a slightly better reduction in NF-jBexpression (Fig. 5d). Also the food supplementsdecreased NF-jB protein levels compared topCTR, in particular FS3 with a reduction byabout 1.2-fold. Instead, COMP-2 levels werereduced withmore efficacy by Ph2, FS2, and FS3;respectively by about 1.3-, 1.5-, and 1.6-fold.

Statistical Analysis The data are presented asthe mean ± SD. When there was no variabilityin the values for a group, a one-sample t test wasused to compare the group mean to that value;otherwise a two-sample t test was used to com-pare group means. p\0.01 was considered sig-nificant in each of the comparative analyses.

DISCUSSION

The biological activity of CS and GlcN foodsupplements could greatly vary according totheir quantity and purity [13, 14], the structuralheterogeneity of the CS (e.g., different molec-ular weight, mixed origin, bound proteins), andmoreover the presence of other components,contaminants, or adulterants [22, 23]. To fill thegap caused by the absence of any specific offi-cial indication, in this study a systematicapproach was designed to analyze at thechemical, biochemical, and biological level 10European FS samples, in comparison with twopharmaceutical grade products. Compared toPh grade CS-based products, most of the FSsamples proved to have high insoluble portions,probably due to the presence of plant extract

residues. Results showed that 7/10 FS presentedinsolubles higher than 25% of the total dryweight. The analyses of FS chromatographicprofiles showed the presence of many species inthe samples, especially unretained molecules,and highlighted a very low representativenessof CS main peaks compared to pharmaceuticalgrade samples. This may be ascribed to lessstandardized and less robust (validated) manu-facturing processes. A recently developedmethod exploiting high-performance anion-exchange chromatography was used to deter-mine both the CS and GlcN contents and toquantify the KS contamination. In the majorityof the analyzed European food supplements,the CS and GlcN contents were lower than thedeclared contents (in 9/10 and 7/10, respec-tively), with over half of them presenting dif-ferences higher than 10% [24, 27]. Followingthe analytical characterization, the samples ofboth Ph and FS were compared for their bio-logical activity by using three human primarycell models: (a) the first based on chondrocytesisolated from nasal cartilage and exposed to IL-1b to mimic osteoarthritis in vitro conditions[30, 31]; (b) the second using articular chon-drocytes; and (c) the third based on synovio-cytes isolated from knee cartilage and synovialfluid of OA-affected patients, respectively [32].The human primary cell models were alreadyused in our laboratory for previous experimentsaimed at evaluating the biological activity ofdifferent sourced CS [30, 33].

Synovial cell cultures exposed to lipopolysac-charide treatment, instead, were reported in theliterature as a model to study the protective andanti-inflammatory effects of HA alone or incombination with CS and GlcN [34, 35]. In addi-tion, through the NF-jB activation and IL-8 pro-duction, the synovial cells have been used toevaluate the anti-inflammatory response of foodsupplements with other potentially bioactivemolecules (e.g., HA, collagen peptides, Boswelliaserrata, devil’s claw, or curcumin). Specifically,NF-jB is a signaling molecule known to play animportant role in the regulation of inflammatorymediators involved in the pathogenesis of OA[36]. We therefore assessed this biomarker in thepathological primary cells, after treatment withPh and/or FS products, finding a significant

bFig. 5 Biological activity on synoviocytes. a Cell picturepanel in the presence of food supplements and pharma-ceutical grade products versus pathological CTR. b Cellviability using trypan blue staining. c IL-6 and IL-8cytokine quantification through ELISA. d Western blot-ting analyses of NF-jB and COMP-2 versus actinhousekeeping protein normalization. T test analyses(*p\ 0.01) were performed to compare the significanceof both pharmaceutical grade CS samples and foodsupplements on IL-6 and IL-8 production, on NF-jBand COMP-2 protein expression by synoviocytes withrespect to pathological non-treated cells

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decrease in Ph1- and Ph2-treated cells, while onlyone of the FS showed a similar positive behavior.Also, COMP-2 is strictly related to OA as a diag-nostic and prognostic indicator of the diseaseseverity and the effect of treatment [33]. The datafrom this experimental research clearly indicatedthat the lower control on the FS compositiondoesnot support their biological efficacy. Specifically,among 10 FS tested only three do not negativelyaffect cell viability and showed efficacy on thereduction of pro-inflammatory cytokines (e.g., IL-6 and IL-8) and on the expression of NF-jB andCOMP-2. On the other hand, the pharmaceuticalgrade products, Ph1 and Ph2, proved to have avery good biological activity. In particular, theysustain cell viability in pathological models andare able to reduce IL-6 and IL-8 production andalso to modulate NF-jB and COMP-2 expression,proving that they have anti-inflammatory prop-erties. Among the food supplements tested onlyFS2 and FS3 proved to be not cytotoxic for all thethree cell-based models, while FS5 and FS6reduced viability of nasal and articular chondro-cytes. FS3 proved to be the best among the FS interms of quality and bioactivity. In fact, it reducedIL-6 expression in all three cellular modelsinvestigated, but decreased IL-8 amounts only innasal chondrocytes and synoviocytes. In vitrocomparative analyses may improve knowledgeon themechanismof action, and also suggest thatthe diverse in vivo outcome, often reported in thescientific literature, may probably be related tothe different product quality administered topatients [37]. In fact, compared to the pharma-ceutical grade products, FS contained low qualityCS, highly contaminated by keratan sulfate, andCS of multiple origins that was often present[23, 27].

Apart from data reported in the literature, allthese additional analytical data perfectly matchwith the biological assay outcomes. Pharma-ceutical grade products proved to be biologi-cally active in all the proposed cellular models,while only three of the 10 screened food sup-plements presented appropriate biologicalactivity. Specifically Ph products significantlyreduced the secreted inflammatory cytokines,through the NF-jB pathway also significantlyreducing COMP-2, considered a biomarker fornegative prognosis on OA progression. These

results support the ESCEO recommendation touse, among all chondroitin products available,the pharmaceutical grade prescription chon-droitin products, for which the evidence base isunequivocal [38].

Limitations of the Current Study

The main limitation is due to the evaluation ofbioactivity in in vitro models, which resemblethe in vivo pathology but are not completelyequivalent to a whole organism (animal,humans). In fact, in vitro tests are only one partof the global picture and pharmacokinet-ics–pharmacodynamics studies as well as thedemonstration of a clinical equivalence/non-inferiority would also be of great interest [38]. Inaddition, the FS studies were from Europeancountries so corresponding American and Asianproducts should be analyzed for a global view ofthe market situation, and the benefit/risk ratiofor patients.

CONCLUSIONS

In this paper a systematic assessment of 10 CS-and GlcN-based food supplements commer-cialized throughout European countries wasperformed and compared to pharmaceutical CS.The results indicated that most of them do notperfectly match the descriptions reported onthe labels in that they present lower CS andGlcN contents. On the other hand, Ph-basedproducts were consistent in purity and titer.Addition of Ph samples elicited beneficial effectsat multiple levels in the three human primarycell models tested in vitro. In contrast, aminority of FS assayed were able to sustain cellviability and to reduce inflammation. Theseresults may help researchers and clinicians toimprove knowledge and understanding whenanalyzing and interpreting clinical outcomes inscientific reports. In general, on the basis ofthese data, only a small percentage of FS prod-ucts containing CS might have similar biologi-cal properties to pharmaceutical grade CS. MostFS containing CS are of uncertain quality withequivocal efficacy and doubtful safety in thetreatment of OA.

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ACKNOWLEDGEMENTS

Funding. This work was partially supportedby PON01_1226 NUTRAFAST, MIUR Ministerodell’Universita e della Ricerca Scientifica. Bio-teknet financed two short-term grants forgraduate technicians. The journal’s Rapid Ser-vice and Open Access fees were funded by IBSACH.

Authorship. All named authors meet theInternational Committee of Medical JournalEditors (ICMJE) criteria for authorship for thisarticle, take responsibility for the integrity ofthe work as a whole, and have given theirapproval for this version to be published.

Disclosures. Antonietta Stellavato, OdileFrancesca Restaino, Valentina Vassallo, RosarioFinamore, Carlo Ruosi, Elisabetta Cassese, MarioDe Rosa, and Chiara Schiraldi have nothing todisclose.

Compliance with Ethics Guidelines. Thisarticle is not based on in vivo studies, on eitheranimals or humans. However, the cells usedwere obtained by digestion of joint/cartilagetissues harvested from surgical procedures onhuman participants. The pieces would havebeen wasted and, as part of a collaborationscheme, these were used instead to set upin vitro models used in this experimentalresearch project. The protocol was approved bythe ethical committee of the University (AOU-SUN) (protocol registration No. 0003711/2015of the 16/05/2015; study title ‘‘Glicosam-minoglicani e/o derivati biotecnologici utili allarigenerazione tessutale’’). Informed consent wasobtained from all participants involved in thisstudy.

Data Availability. All relevant data arereported within the manuscript. All relevantdata are presented in the results section andonly those are discussed in the discussionsection.

Open Access. This article is distributedunder the terms of the Creative Commons

Attribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any non-commercial use, distribution, and reproductionin any medium, provided you give appropriatecredit to the original author(s) and the source,provide a link to the Creative Commons license,and indicate if changes were made.

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