The Open Orthopaedics Journal




ISSN: 1874-3250 ― Volume 13, 2019

The Role of Different Hyaluronic Acids in the Articular Cartilage of Rabbit



Jaime Antonio Sánchez Lázaro*, Pilar Coronel Granado, Mercedes Gimeno del Sol, Ana González Medina, Luis Díaz Gállego, Daniel González-Arabio Sandoval, Julio Gabriel Prieto Fernández
1 Departamento de Ciencias Biomédicas, Facultad de Veterinaria, University of León, Campus de Vegazana s/n, León 24071, León, Spain

Abstract

Purpose:

To elucidate if the differences found in the physico-chemical and rheological behaviour of Hyaluronic Acids result in different in vivo activity. For this purpose two Hyaluronic Acids (HA), HA-1 and HA-2, with similar molecular weight but different percentage of concentration variation, were compared through an osteoarthritis model.

Methods and Materials:

Osteoarthritis was induced in white New Zealand rabbits by anterior cruciate ligament section. After the induction period, the animals were allocated to receive HA-1 or HA-2 intra-articularly in one knee whereas the contralateral knee was used as Operated Control. An additional group of non-operated animals was used as Healthy Controls. Samples of cartilage were taken for different measures: apoptosis, nitric oxide (nitrites) and hyaluronic acid in synovial fluid.

Results:

The administration of HA-1 had a significant inhibitor effect on apoptosis of the chondrocytes compared to operated untreated animals (p = 0.0089), whereas this difference was not observed in the HA-2 knees. Levels of nitrites determined by HPLC in the HA-1 knees were similar to those in the Healthy group (p = 0.6551) whereas they were significantly higher in Operated Control and HA-2 groups (p = 0.0001). The comparison between HA-1 and HA-2 also revealed significantly lower levels of nitrites in the HA-1 knees (p = 0.0001). Values of hyaluronic acid in synovial fluid did not show statistical differences between the different study groups.

Conclusions:

HA-1 and HA-2 showed different physico-chemical characteristics and these differences have resulted in different in vivo behaviour. As a consequence, not all the HA with similar molecular weight can be considered as equivalent.

Keywords: Hyaluronic acid, osteoarthritis, cytometry, TUNEL, HPLC..


Article Information


Identifiers and Pagination:

Year: 2010
Volume: 4
First Page: 44
Last Page: 47
Publisher Id: TOORTHJ-4-44
DOI: 10.2174/1874325001004010044

Article History:

Received Date: 11/11/2009
Revision Received Date: 24/11/2009
Acceptance Date: 3/10/2009
Electronic publication date: 19/1/2010
Collection year: 2010

Article Metrics:

CrossRef Citations:
0

Total Statistics:

Full-Text HTML Views: 1793
Abstract HTML Views: 1049
PDF Downloads: 300
Total Views/Downloads: 3142

Unique Statistics:

Full-Text HTML Views: 853
Abstract HTML Views: 647
PDF Downloads: 187
Total Views/Downloads: 1687
Geographical View

© Sánchez Lázaro et al.; Licensee Bentham Open.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/) which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.


* Address correspondence to this author at the Departamento de Ciencias Biomédicas, Facultad de Veterinaria, University of León, Campus de Vegazana s/n, León 24071, León, Spain; Tel: 0034-987291263; Fax: 0034-987291267; E-mail: jasanlazaro@telefonica.net





INTRODUCTION

Hyaluronic Acid (HA) is responsible for the viscoelastic properties of synovial fluid, which is greatly diminished in osteoarthritic joints in both concentration and molecular weight [1Balazs EA, Laurent TC, Jeanloz RW. Nomenclature of the hyaluronic acid Biochem J 1986; 235(3): 903.]. The main objective of intra-articular treatment with HA is to restore the viscoelastic properties of the synovial fluid. In a recent review of existing treatment guidelines, HA is recommended for the treatment of knee OA (level of evidence Ia) [2Zhang W, Moskowitz RW, Nuki G, et al. OARSI recommendations for the management of hip and knee osteoarthritis, part I: Critical appraisal of existing treatment guidelines and systematic review of current research evidence Osteoarthr Cartil 2007; 15: 981-1000.]. It has been demonstrated that HA reduces osteoarthritic pain owing to a reduction of nociceptive and sensorial response in the articular pain receptors [3Pozo MA, Balazs EA, Belmonte C. Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronic derivative Exp Brain Res 1997; 116(5): 3-9.]. In the same way, in vitro studies [4Yoshioka M, Shimizu C, Harwood FL, Coutts RD, Amiel D. The effects of hyaluronan during the development of osteoarthritis Osteoarthr Cartil 1997; 5: 257-60., 5Shimizu C, Yoshioka M, Coutts Rd, et al. Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee Osteoarthr Cartil 1998; 6: 1-9.] have demonstrated the protective effects of HA on the cartilage in experimental models of osteoarthritis. Other in vitro studies have also demonstrated that HA has beneficial effects on the extra-cellular matrix [6Frean SP, Abraham LA, Lees P. In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen Res Vet Sci 1997; 67: 183-90.], the immune system cells [7Balazs EA, Briller S, Denlinger JL. Na-hyaluronate molecular size variations in equine and human arthritic synovial fluids and the effect on phagocycit cells Semin Arthritis Rheum 1981; 11: 141-3.] and inflammation mediators [8Takahashi K, Goomer RS, Harwood F, Kubo T, Hirasawa Y, Amiel D. The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1beta (IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis Osteoarthr Cartill 1999; 7: 182-90.]. There are several preparations of HA authorized for the treatment of osteoarthritis from different sources, manufacturing process, molecular weight and final concentration. A recent study [9Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9.] made it clear that the rheological properties, the molecular weight of HA and its concentration are factors which could determine the intra-articular viability of the molecule. Mechanical and chemical degradation were both reduced in HAs with low molecular weight, with the final concentration of the product being a critical factor. Considering the differences found, and to elucidate whether the different physico-chemical behaviour had, as a consequence, different in vivo activity, we decided to conduct the present study. The comparison is made using an experimental model of osteoarthritis developed by our team [10Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76.].

MATERIALS AND METHODS

Experimental Model

Osteoarthritis was induced in white New Zealand rabbits kept at the animal facilities of University of Leon, Spain. The study was performed in accordance with the laws in force relative to the protection of animals used for experimentation and other scientific purposes and was favourably informed by the Ethics Commission of the University of León. Postoperatively, the rabbits were housed in a cage, after which they were allowed unlimited activity. During follow-up no joint immobilization was used.

Hyaluronic Acid

Two Hyaluronic Acids were studied, both have similar source (biofermentation), mechanical and optical properties, but one revealed fluctuating concentrations in a prior study (Prieto JG et al. HA-2 has a 15% concentration variation (8.39 ± 1.65 mgml-1) while other HA studied (including HA-1) have a 7% variation. HA-1 (900 KDa) (AdantÒ Tedec-Meiji Farma, Spain) and HA-2 (1200 KDa) (OstenilÒ TRB Chemedica AG, Germany). Depolimerisation processes is highly dependent on the sample concentration, HA-2 results in higher depolymerisation than HA-1 with tetracyclines and thermal depolymerisation [9Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9.].

Osteoarthritis Induction

Anterior Cruciate Ligament (ACL) section was performed, based on the Pond-Nuki model [11Pond MJ, Nuki G. Experimentally induced osteoarthritis in the dog Ann Rheum Dis 1973; 32: 387-8.], under local anaesthesia by percutaneous surgery through a 2-3 mm incision using a curved scalpel (Cutfix Stitch Cutter, B. Braun Surgical GMBH) [10Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76.].

Study Design

In total 36 animals were used and the ACL of both knees were sectioned in all of them. After ten weeks’ development of osteoarthritis, the animals were randomly assigned 1:1 to receive HA1 or HA2. HA was injected intra-articularly into one knee for three consecutive weeks at a dose of 0.1 ml/kg weight (0.3 ml/knee, in rabbits with an average weight of 3kg) one injection each week, leaving the other knee to be used as Operated Control. The animals were sacrificed two weeks after the last HA injection was administered. Control Group: Four untreated animals of the same age as the operated animals, not having undergone surgery were used as Healthy Control (8 control knees).

After a 500µl saline injection, samples of synovial fluid were taken from all knees, where possible, so as to quantify the HA molecular weight and concentration by High Performance Liquid Chromatography (HPLC) [9Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9., 10Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76., 12Tulamo RM, Houttu J, Tupamaki A, Salonen M. Hyaluronate and large molecular weight proteoglycans in synovial fluid from horses with various arthritides Am J Vet Res 1993; 57: 932-7.-14Coleman PJ, Scott D, Ray J, Mason RM, Levick JR. Hyaluronan secretion into synovial cavity of rabbit knees and coimpararison with albumin turnover J Physiol 1997; 503: 645-56.]. Gross morphological changes to the femoral condyles, trochlea and tibial surfaces in the rabbits were assessed during the collection of cartilage samples according to the method and the grading scale reported by Yoshiaka et al. [15Yoshioka M, Coutts RD, Amiel D, Hacker SA. Characterization of a model of osteoarthritis in the rabbit knee Osteoarthr Cartil 1996; 4: 87-98.]. The entire surface of both femoral condyles (internal and external to obtain a greater number of chondrocytes) was taken in order to measure apoptosis by TUNEL and cytometry. TUNEL method as a Gold Standard method [16Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology Arthritis Rheum 1998; 41: 284-9.-19Labat-Moleur F, Guillermet C, Lorimier P, et al. TUNEL apoptotic cell detection in tissue sections: Critical evaluation and improvement critical evaluation and improvement J Histochem Cytochem 1998; 46: 327-4.] was done after initial fixation of the samples with 10% formaldehyde, paraffin embedded cut into sections which were processed (ApopTag® Plus peroxidase in situ Apoptosis Detection Kit. Chemicon International, USA), cell in apoptosis were quantified by an external department and evaluated blindy. Cytometry with Annexin Kit (BD Pharmingen, San Jose, CA USA), after 6 hours cartilage hyaluronidase (Sigma H-3884; 0.1mg/ml) and IIS-type collagenase (C-1764; 2mg/ml) digestion [20Jakob M, Demarteau O, Schafer D, Stumm M, Heberer M, Martin I. Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells Connect Tissue Res 2003; 44: 179-80.-22Takahashi K, Hashimoto S, Kubo T, Hirasawa Y, Lotz M, Amiel A. Effect of hyaluronan on chondrocyte apoptosis and nitric oxide production in experimentally induced osteoarthritis J Rheumatol 2000; 27: 1713-20.]. Cartilage from the tibial plateau was used for the indirect quantification of nitric oxide in cultured cartilage using the Griess method. The supernatant of cartilage digestion for cytometry study was used for nitrites and nitrates studies by HPLC [23Tsikas D, Gutzki FM, Stichtenoth DO. Circulating and excfretory nitrite and nitrate as indicator of nitric oxide synthesis in humans: Methods of analysis Eur J Clin Pharmacol 2006; 62: 51-9., 24Jobgen WS, Jobgen SC, Li H, Meininger CJ, Wu G. Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography J Chromatogr 2007; 851: 71-82.].

Statistical Analysis of Data

The Mann-Whitney Test was used to analyse the results of gross morphology. All the remaining parameters studied were analysed using ANOVA–MANOVA with the Newman-Keuls Test. Statistical significance was set at p < 0.05.

RESULTS

Gross Morphology (Fig. 1)

Fig (1)

*Grade III and IV: Operated Control vs control (p = 0.002), Operated Control vs treated groups (p = 0.003).



When considering the most severe grades (III and IV) altogether, statistically significant differences were found between Operated Contol group and both Control (p = 0.002) and Treated groups (HA1 or HA2) (p = 0.003). No differences were found between Control and Treated groups (p = 0.185).

Levels of Apoptosis and Necrosis (Cytometry) (%) (Fig. 2).

Fig (2)

*Operated Control, HA2 and HA1 groups versus Control (p = 0.0018, 0.0001, 0.0001), # HA1 versus Operated Control group (p = 0.0089).



Apoptosis grade. TUNEL method (%) (Fig. 3).

Fig (3)

No statistically significant differences were found in any of the comparisons made between groups.



Nitrites and nitrates in supernatant (µg/ml) (HPLC) (Fig. 4).

Fig (4)

*Operated Control and Ha2 versus Control (p = 0.0001, 0.0001), ♦ Operated Control versus Ha2 (p = 0.0248), # HA1 versus Operated Control and HA2 (p = 0.0001, 0.0001), HA1 versus control (p = 0.5245).



Hyaluronic Acid in Synovial Fluid (HPLC) (Fig. 5).

Fig (5)

No statistically significant differences were found in any of the comparisons made between groups.



DISCUSSION

The administration of HA1 for three weeks in rabbits after ten weeks osteoarthritis-induction, had an inhibiting effect on the apoptosis of the chondrocytes compared to the operated Control group (cytometry study), with statistically significant differences (p = 0.0089) similar to that observed in a five week study [10Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76.]. However, this difference was not reached in the group treated with HA2 (p = 0.0696). No differences between either treatment groups was found (p = 0.2176). The study of necrosis did not reveal statistically significant differences between the different study groups. The apoptosis levels determined by TUNEL were clearly higher in the Operated Control group, although the high standard deviation values made the differences not statistically significant. This method has been, up to now, considered the Gold Standard [16Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology Arthritis Rheum 1998; 41: 284-9., 25Borderie D, Hilliquin P, Hernvann A, Lemarechal H, Menkes CJ, Ekindjian OG. Apoptosis induced by nitric oxide is associated with nuclear p53 protein expression in cultured osteoarthritic synoviocytes Osteoarthritis Cartilage 1999; 7: 203-13., 26Nishida K, Doi T, Matsuo M, et al. Involvement of nitric oxide in chondrocyte cell death in chondro-osteophyte formation Osteoarthr Cartil 2001; 9: 232-7.]. Nevertheless its variability has been questioned in recent investigations. A new method to replace the TUNEL method for another with better possibilities of quantification (for instance chondropoptosis determined by caspase-2L or caspase-3) must therefore be considered for future studies [27Perez HE, Luna MJ, Rojas ML, Kouri JB. Chondroptosis: an immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage Apoptosis 2005; 10: 1105-0.]. Gross morphological study did not revealed differences, the advantage of this method regarding a complete visual histological assessment scale (ICRS) [28Mainil-Varlet P, Aigner T, Brittberg M, et al. Histological assessment of cartilage repair: A report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J Bone Joint Surg Am 2003; 85-A(2): 45-57.], is that in small experimental animals as rabbits, valuable samples are not lost they can be employed in other interesting study methods as flow cytometry. In the near future probably immunohistochemical staining should further displace conventional histological techniques [28Mainil-Varlet P, Aigner T, Brittberg M, et al. Histological assessment of cartilage repair: A report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J Bone Joint Surg Am 2003; 85-A(2): 45-57.].

Levels of nitrites in the knees of both groups treated were significantly lower than those in the Operated Control group. Nevertheless, levels in the animals treated with HA1 were very similar to those obtained in the Control group (p = 0.6551), whereas those treated with HA2 were not (p = 0.0001). This was confirmed by the comparison between HA1 and HA2 that revealed significantly lower levels of nitrites in the HA1 group (p = 0.0001). Levels of nitrates were five to eight times lower than nitrites and follow the same pattern of distribution in the different groups; nevertheless, there were no statistically significant differences, probably due to the high standard deviation values found. The differences found when analysing nitrites are maintained when both nitrites and nitrates are considered together as stable metabolites of nitric oxide. Values of HA in synovial fluid did not show any statistical differences between the different study groups. The molecular weight of the HA in the synovial fluid of the operated Control knees was 17% lower than in the control group. The groups treated showed less reduction of HA molecular weight (10% HA2, 13% HA1) as could be expected after the administration of exogenous HA and in accordance with the molecular weights of HA1 and HA2, respectively. According to a consensus reached at the 7th International Conference on hyaluronan (South California, 2007) both HAs are included as HAs of medium molecular weight, and both have potentially disease-modifying effects [29Yatabe T, Mochizuki S, Takizawa M, et al. Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes Ann Rheum Dis 2009; 68(6): 1051-8.] as inhibition IL-1β stimulated production of MMP-1, MMP-3 and MMP-13 [30Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T. Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage Arthritis Rheum 2004; 50(2): 516-25.].

Considering the results shown above, it seems to be clear that in vivo activity is not influenced by molecular weight. Recent studies published by our group [9Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9.] stated that considering the elasto-viscous nature of HA, not only molecular weight but stability against degradation processes by mechanical and chemical factors, is an important factor. Rheological properties and final concentration are also critical aspects to be taken into account in the final behaviour of the molecule when injected into the articular space. In the study, differences were also found among the HAs of similar molecular weight, especially with HA2 where the fluctuating concentrations observed led to lower stability results than expected [9Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9.]. In this study, the animals treated with HA1 not only showed statistically significant lower NO levels regarding untreated animals, but the values were also similar to the ones in the healthy Control group. The animals treated with HA2 showed lower NO values than the untreated animals, but levels were much higher than the control group (statistically significant). In addition the comparison between treatment groups, HA1 and HA2, revealed significantly lower levels of nitrites in the HA1 group. It is known that NO is able to induce apoptosis in chondrocytes and that, moreover, osteoarthritic cartilage contains a higher percentage of cells undergoing apoptosis than normal cartilage [31Conrozier TH. Death of articular chondrocytes. Mechanism and protection Press Med 1998; 27: 1859-61.]. In conclusion, the protective role of HA1 on the osteoarthritic cartilage is also confirmed with similar results to those obtained in previous studies [10Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76.]. HA1 and HA2 showed different physico-chemical characteristics and these differences have resulted in different in vivo behaviour. Consequently, the two products cannot be considered as equivalent. HA1 has shown higher effects on inflammation processes than HA2, which could probably provide better results and a long-term protective effect. Along the same line of research, a long-term clinical trial is under development to confirm this protective action in humans, and a new study focus on extracellular matrix is required to confirm differences founded in this study.

CONFLICT OF INTEREST STATEMENT

Part of this study has been supported by Tedec-Meiji Farma as a project integrated in the University-Industry collaboration program issued by the University of León (Spain). As an university project no economical benefits in any form have been received or will be received from a commercial party directly or indirectly to the subject of this article by the authors. Our group has been working in Hyaluronic Acid projects with this University-Industry plan with almost all Hyaluronic Acid companies including TRB Chemedica AG.

ACKNOWLEDGEMENTS

We would like to thank Claire Byrne for her contribution in the translation of the manuscript.

This study was supported by a grant from the University-Industry agreement plan.

REFERENCES

[1] Balazs EA, Laurent TC, Jeanloz RW. Nomenclature of the hyaluronic acid Biochem J 1986; 235(3): 903.
[2] Zhang W, Moskowitz RW, Nuki G, et al. OARSI recommendations for the management of hip and knee osteoarthritis, part I: Critical appraisal of existing treatment guidelines and systematic review of current research evidence Osteoarthr Cartil 2007; 15: 981-1000.
[3] Pozo MA, Balazs EA, Belmonte C. Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronic derivative Exp Brain Res 1997; 116(5): 3-9.
[4] Yoshioka M, Shimizu C, Harwood FL, Coutts RD, Amiel D. The effects of hyaluronan during the development of osteoarthritis Osteoarthr Cartil 1997; 5: 257-60.
[5] Shimizu C, Yoshioka M, Coutts Rd, et al. Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee Osteoarthr Cartil 1998; 6: 1-9.
[6] Frean SP, Abraham LA, Lees P. In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen Res Vet Sci 1997; 67: 183-90.
[7] Balazs EA, Briller S, Denlinger JL. Na-hyaluronate molecular size variations in equine and human arthritic synovial fluids and the effect on phagocycit cells Semin Arthritis Rheum 1981; 11: 141-3.
[8] Takahashi K, Goomer RS, Harwood F, Kubo T, Hirasawa Y, Amiel D. The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1beta (IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis Osteoarthr Cartill 1999; 7: 182-90.
[9] Prieto JG, Pulido MM, Zapico J, et al. Comparative study of hyaluronic derivatives: Rheological behaviour, mechanical and chemical degradation. Int J Biol Macromol 2005; 35: 63-9.
[10] Díaz-Gallego L, Prieto JG, Coronel P, Gamazo LE, Gimeno M, Alvarez AI. Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment J Orthop Res 2005; 23: 1370-76.
[11] Pond MJ, Nuki G. Experimentally induced osteoarthritis in the dog Ann Rheum Dis 1973; 32: 387-8.
[12] Tulamo RM, Houttu J, Tupamaki A, Salonen M. Hyaluronate and large molecular weight proteoglycans in synovial fluid from horses with various arthritides Am J Vet Res 1993; 57: 932-7.
[13] Coleman PJ, Scott D, Mason RM, Levick JR. Characterization of the effect of high molecular weight hyaluronan on trans-synovial flow in rabbit knees J Physiol 1999; 514: 265-82.
[14] Coleman PJ, Scott D, Ray J, Mason RM, Levick JR. Hyaluronan secretion into synovial cavity of rabbit knees and coimpararison with albumin turnover J Physiol 1997; 503: 645-56.
[15] Yoshioka M, Coutts RD, Amiel D, Hacker SA. Characterization of a model of osteoarthritis in the rabbit knee Osteoarthr Cartil 1996; 4: 87-98.
[16] Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology Arthritis Rheum 1998; 41: 284-9.
[17] Gold R, Schmied M, Giegerich G, et al. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques Lab Invest 1994; 71: 219-5.
[18] Negoescu A, Lorimier P, Labat-Moleur F, et al. In situ apoptotic cell labelling by the TUNEL method: Improvement and evaluation on cell preparations J Histochem Cytochem 1996; 44: 959-68.
[19] Labat-Moleur F, Guillermet C, Lorimier P, et al. TUNEL apoptotic cell detection in tissue sections: Critical evaluation and improvement critical evaluation and improvement J Histochem Cytochem 1998; 46: 327-4.
[20] Jakob M, Demarteau O, Schafer D, Stumm M, Heberer M, Martin I. Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells Connect Tissue Res 2003; 44: 179-80.
[21] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis Semin Hematol 2001; 38: 179-93.
[22] Takahashi K, Hashimoto S, Kubo T, Hirasawa Y, Lotz M, Amiel A. Effect of hyaluronan on chondrocyte apoptosis and nitric oxide production in experimentally induced osteoarthritis J Rheumatol 2000; 27: 1713-20.
[23] Tsikas D, Gutzki FM, Stichtenoth DO. Circulating and excfretory nitrite and nitrate as indicator of nitric oxide synthesis in humans: Methods of analysis Eur J Clin Pharmacol 2006; 62: 51-9.
[24] Jobgen WS, Jobgen SC, Li H, Meininger CJ, Wu G. Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography J Chromatogr 2007; 851: 71-82.
[25] Borderie D, Hilliquin P, Hernvann A, Lemarechal H, Menkes CJ, Ekindjian OG. Apoptosis induced by nitric oxide is associated with nuclear p53 protein expression in cultured osteoarthritic synoviocytes Osteoarthritis Cartilage 1999; 7: 203-13.
[26] Nishida K, Doi T, Matsuo M, et al. Involvement of nitric oxide in chondrocyte cell death in chondro-osteophyte formation Osteoarthr Cartil 2001; 9: 232-7.
[27] Perez HE, Luna MJ, Rojas ML, Kouri JB. Chondroptosis: an immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage Apoptosis 2005; 10: 1105-0.
[28] Mainil-Varlet P, Aigner T, Brittberg M, et al. Histological assessment of cartilage repair: A report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J Bone Joint Surg Am 2003; 85-A(2): 45-57.
[29] Yatabe T, Mochizuki S, Takizawa M, et al. Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes Ann Rheum Dis 2009; 68(6): 1051-8.
[30] Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T. Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage Arthritis Rheum 2004; 50(2): 516-25.
[31] Conrozier TH. Death of articular chondrocytes. Mechanism and protection Press Med 1998; 27: 1859-61.

Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


Browse Contents




Webmaster Contact: info@benthamopen.net
Copyright © 2019 Bentham Open