273

http://journals.tubitak.gov.tr/veterinary/

Turkish Journal of Veterinary and Animal Sciences Turk J Vet Anim Sci(2017) 41: 273-281© TÜBİTAKdoi:10.3906/vet-1605-107

The comparison of the effects of intraarticular injections of bovine amnioticfluid and hyaluronic acid on cartilage tissue in an experimental osteoarthritic

rabbit model: histopathological and immunohistochemical results

Murat TANRISEVER1,*, Hatice ERÖKSÜZ2, Sait BULUT1

1Department of Surgery, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey2Department of Pathology, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey

* Correspondence: mtanrisever@firat.edu.tr

1. IntroductionOsteoarthritis (OA) is a degenerative joint disease that affects the synovial joints and is characterized by degradation and loss of articular cartilage with subchondral bone remodeling, osteophyte formation, and inflammation of the synovium. Clinical signs include joint pain, stiffness, swelling, and loss of mobility, increasing in severity as the disease progresses (1,2).

To date, no definitive treatment method has been found for OA. Studies are underway to reduce progressive cartilage damage, which is the basic pathology of the disease.

The cartilage healing effects of hyaluronic acid (HA) and its effects on slowing the progression of OA are known and it is widely used today in the treatment of OA (3–6).

In addition, human amniotic fluid has been used as an alternative to HA in the prevention of cartilage damage in OA and in cartilage healing in a few studies (7–9). The bovine amniotic fluid (BAF) that we use in this study contains insulin-like growth factors that have a stimulating effect on mesenchymal cells and chondrocytes and other growth factors in addition to macromolecules such as

HA and HA-activating agent (10–13). It also contains metalloproteinase inhibitors that can prevent cartilage destruction (14).

While the mechanism is not yet clarified, there is a positive correlation between cartilage damage and apoptosis (15,16). Similar studies supporting the same view have demonstrated that caspases (cysteinyl aspartate-specific proteinases) such as caspase-3 and caspase-8 are increased in OA patients (17). With the effect of proteolytic enzymes that cause collagen disintegration, OA causes matrix loss in the joint cartilage. MMP-13 is an important matrix metalloproteinase in OA and is expressed significantly in cartilage tissue due to type II collagen disintegration (18,19).

The aim of this report was investigate the efficacy of BAF, which is much easier to access and more economical, on cartilage tissue in OA based on the histopathological and immunohistochemical (caspase-3 and caspase-8 expression in chondrocytes and MMP-13 expression in cartilage matrix) investigation of its effects as compared with the widely used HA.

Abstract: In this experimental study, effects of bovine amniotic fluid and hyaluronic acid were compared on an osteoarthritic model induced in rabbit stifle joints. The study was performed on 21 rabbits undergoing anterior cruciate ligament transaction for establishing osteoarthritis. Four weeks later, the rabbits were divided into 3 equal groups of 7 animals each. The first group of rabbits received intraarticular injection of 0.5 mL of bovine amniotic fluid (BAF) three times at 1-week intervals. The second group of rabbits received intraarticular injection of 0.5 mL of hyaluronic acid (HA) three times at 1-week intervals. The third group of rabbits served as a control with no application. Articular surfaces of the femur and tibia were evaluated using the Mankin scoring system for histopathological cartilage examination and were evaluated for immunohistochemical examination by using caspase-3, caspase-8, and MMP-13. All statistical results showed that there were highly significant differences between the control group and the other groups. On the other hand, there was no significant difference between the BAF and HA groups (P < 0.01). It was concluded that the use of BAF, which is an inert fluid rich in growth factors, had protective effects against degenerative cartilage changes in an experimental rabbit model and can be an alternative agent in cartilage protective therapy.

Key words: Experimental osteoarthritis, hyaluronic acid, bovine amniotic fluid, caspase-3, caspase-8, MMP-13

Received: 30.05.2016 Accepted/Published Online: 19.10.2016 Final Version: 19.04.2017

Research Article

274

TANRISEVER et al. / Turk J Vet Anim Sci

2. Materials and methodsThis study was conducted with the approval (number 46 dated 04.04.2013) of the Fırat University Rectorate Animal Experiments Ethics Board Presidency. The experimental animals were obtained from the Fırat University Experimental Research Center (FUERC). The study was conducted at the FUERC and the Fırat University Animal Hospital Small Animal Clinic operating rooms.

Twenty-one male and female New Zealand rabbits of 5 months old with an average weight of 2700–3000 g were used in the study.

In the literature there are 3 different types of experimental animal studies OA models:1. Genetically manipulated spontaneous or naturally

occurring models, 2. Mechanical models,3. Chemical models.

Today the most widely used approach creates mechanical instability OA models that are similar to the chronic traumatic model (20). The most commonly preferred mechanical surgical applications in animal OA models are partial meniscectomy (21), complete meniscectomy (22), anterior cruciate ligament transection (20), anterior cruciate ligament transection with partial meniscectomy (23), anterior and posterior cruciate ligaments transection, varus-valgus osteotomy, and extraarticular surgery procedures such as muscle resection (20). In this study, the method of cutting the anterior cruciate ligaments was employed in order to create experimental OA in the rabbits.

The rabbits were anesthetized with 5 mg/kg xylazine hydrochloride (Rompun, 23.32 mg/mL, Bayer, İstanbul,

Turkey) and 35 mg/kg ketamine hydrochloride (Ketalar, 50 mg/mL, Eczacıbaşı, İstanbul, Turkey) intramuscularly.

After anesthesia, the right knees of the rabbits were shaved and they were laid down in the supine position. Before the incision, the knees were sterilized with 10% povidone iodine (Batticon, Adeka, Samsun, Turkey). Then, approaching with a lateroanterior longitudinal incision, the patella was deviated to the medial following lateral parapatellar arthrotomy and the anterior cruciate ligament was cut. After whether the cruciate ligament had been completely cut was evaluated using the anterior drawer test, the patellar tendon and skin of the test subjects were closed with separate sutures using polyglactin 2/0 suture material (Vicryl, Johnson & Johnson Medical, Brussels, Belgium) (Figure 1). Antibiotics (İecilline, 400,000 IU, İ.E. Ulagay, İstanbul, Turkey) were given intramuscularly at a vial per day for 5 days.

The experimental animals were allowed normal cage activity after the operation. Four weeks after the surgical intervention, the rabbits were randomly divided into 3 equal groups. The first group of rabbits (n = 7) was given 0.5 mL of intraarticular BAF injections 3 times with 1-week intervals. The second group of rabbits (n = 7) was given 0.5 mL of intraarticular HA applications 3 times with 1-week intervals. The third group of rabbits (n = 7) was used as a control group (CONT) and nothing was applied to them.

The BAF was taken postmortem using injectors from a healthy cow approximately 5–6 months pregnant under aseptic conditions from a slaughterhouse. It was brought to the laboratory without breaking the cold chain using an ice box and kept at –20 °C in deep freeze. It was allowed to dissolve by waiting for 15 min at room temperature before use.

Figure 1. Images of the operation site related to the cutting of the anterior cruciate ligament.

275

TANRISEVER et al. / Turk J Vet Anim Sci

Sodium hyaluronate in a ready-to-use glass injector (Ostenil, 10 mg/mL, Bio-Gen, Ankara, Turkey) was used as HA.

Twelve weeks after the last intraarticular injection was given, all the rabbits were euthanized with ketamine (100 mg/kg, intramuscularly).

Condylus samples including the joint cartilages of the tibia and femur bones taken from the animals that were euthanized after the experiment were identified in 10% formaldehyde and routinely followed after the decalcification process; paraffin blocks were prepared and the sections were stained using the hematoxylin-eosin and safranin O methods. The Mankin scoring system (Table 1) was applied to the preparations in histopathological evaluation. The tissues were immunohistochemically examined with respect to the expression of caspase-3 and caspase-8, indicative of cell apoptosis, and the expression of MMP-13, which is an indicator of collagen destruction and osteoarthritis. The immunohistochemical staining protocol was applied according to the protocol of the commercial kit (Invitrogen Superpicture 3rd Gen IHC Detection Kit, USA). Caspase-3, Ab.4 Rabbit Pab (Neomarkers, ready to use), caspase-8 FLIP antibody, Ab.4 Rabbit Pab (Biorbyt, 1/100-1/200 dilution), and

MMP-13, Ab.1 Mouse Mab (Neomarkers, ready to use) antibodies were used as primary antibodies and DAB (diaminobenzidine) was used as a chromogen.

Statistical analysis of the results was done using SPSS 22. After assessing whether there was a difference between the groups using the Kruskal–Wallis analysis method, the Mankin scores and caspase-3, caspase-8, and MMP-13 expressions were evaluated between the groups as a percentage using the Mann–Whitney U test in order to determine from which group the difference stemmed and whether it was a significant difference or not.

3. ResultsMild lameness was observed in rabbits following the operations for 3–5 days. While in the control group rabbits were observed with symptoms of lameness in the right limbs depending on the progress of osteoarthritis on days 90–120, there were no similar symptoms in rabbits in the BAF and HA groups. 3.1. Macroscopic findings3.1.1. BAF group When the femoral condyles of the BAF group were examined macroscopically, superficial irregularities were seen in 3 cases, pannus formation with surface

Table 1. Mankin scoring system (25).

Category Subsection Grade

Structure

Normal 0

Surface irregularity 1

Pannus and surface irregularity 2

Clefts to transitional zone 3

Clefts to radial zone 4

Clefts to calcified zone 5

Complete disorganization 6

Chondrocyte cell count

Normal 0

Diffuse hypercellularity 1

Clusters 2

Hypocellularity 3

Safranin O staining

Normal 0

Slight reduction 1

Modest reduction 2

Severe reduction 3

No dye noted 4

Tidemark integrityIntact 0

Crossed by blood vessels 1

Total 14

276

TANRISEVER et al. / Turk J Vet Anim Sci

irregularities in 2 cases, and clefts to the transitional zone in 1 case, while the joint surface in 1 case was normal. When the tibial articular surfaces were analyzed, pannus formation with surface irregularity was observed in 1 case, and surface irregularities were observed in 6 cases (Figure 2).3.1.2. HA groupWhen the femoral condyles of the HA group were considered, superficial irregularities were seen in 4 cases and pannus formations with superficial irregularities were observed in 3 cases. When the same group’s tibial articular surfaces were examined, pannus formation in addition to surface irregularity was observed in 1 case, and surface irregularities were observed in 6 cases (Figure 3).

3.1.3. CONT groupWhen the femoral condyles of the CONT group were examined, clefts to the transitional zone were seen in 3 cases, clefts to the radial zone in 3 cases, clefts to the calcified zone in 1 case, and complete disorganization in 1 case. When the same group’s tibial articular surfaces were considered, pannus formation in addition to surface irregularity was observed in 2 cases, clefts to the transitional zone in 4 cases, and clefts to the calcified zone in 1 case (Figure 4).3.2. Microscopic findingsThe femoral and tibial joint surfaces of the right knees of the rabbits were microscopically examined with regard to the degree to which the damage that occurred affected

Figure 2. Macroscopic view of the knee joint of the BAF group.

Figure 3. Macroscopic view of the knee joint of the HA group.

277

TANRISEVER et al. / Turk J Vet Anim Sci

the cartilage layers, the state of the chondrocytes, and the degrees of staining of the matrix with safranin O and tidemark integrity (Tables 2 and 3; Figure 5).3.2.1. Histopathologic findings on the femoral articular surfaceWhen the chondrocyte cells of the BAF group were examined, there was diffuse hypercellularity in 3 cases and cell clusters in 3 cases, and 1 case was normal. When evaluated in terms of staining of the extracellular matrix with safranin O, there were slight reductions in 2 cases, modest reductions in 4 cases, and severe reduction in 1 case. In 2 cases of this group it was seen that tidemark integrity was broken down by vessels.

When the chondrocyte cells of the HA group were examined, there was diffuse hypercellularity in 2 cases, cell clusters in 2 cases, and hypocellularity in 2 cases and 1 case was normal. When evaluated in terms of staining of the extracellular matrix with safranin O, there were slight reductions in 3 cases, modest reductions in 3 cases, and severe reduction in 1 case. In this group there was one case of tidemark integrity broken down by vessels.

When the chondrocyte cells of the CONT group were examined, there were cell clusters in 1 case and hypocellularity in 6 cases. When evaluated in terms of staining of the extracellular matrix with safranin O, there was modest reduction in 1 case, severe reduction in 3

Figure 4. Macroscopic view of the knee joint of the CONT group (general disorganization).

Table 2. Total Mankin scores for each subject’s femoral joint cartilage.

Rabbits BAF HA CONT

1 3 7 14

2 5 5 14

3 5 3 11

4 7 5 12

5 4 8 11

6 9 2 11

7 1 5 9

Total 34 35 82

Average 4.85 5.00 11.71

278

TANRISEVER et al. / Turk J Vet Anim Sci

Figure 5. The hematoxylin and eosin (H.E) and safranin O staining of the joint cartilages of the subjects and immunohistochemical caspase-3, caspase-8, and MMP-13 expressions.

279

TANRISEVER et al. / Turk J Vet Anim Sci

cases, and no dye noted in 3 cases. Tidemark integrity was broken in all of the individuals in this group.3.2.2. Histopathologic findings on the tibial articular surfaceWhen chondrocyte cells of the BAF group were examined, there was diffuse hypercellularity in 1 case and cell clusters in 5 cases, and 1 case was normal. When evaluated in terms of staining of the extracellular matrix with safranin O, there were slight reductions in 2 cases, modest reductions in 3 cases, and severe reductions in 2 cases. In this group there were 2 cases of tidemark integrity broken down by vessels.

When the chondrocyte cells of the HA group were examined, there was diffuse hypercellularity in 3 cases, cell clusters in 3 cases, and hypocellularity in 1 case. When evaluated in terms of staining of the extracellular matrix with safranin O, there were slight reductions in 3 cases, modest reductions in 3 cases, and severe reduction in 1 case. In this group there were 4 cases of tidemark integrity broken down by vessels.

When the chondrocyte cells of the CONT group were examined, there were cell clusters in 4 cases and hypocellularity in 3 cases. When evaluated in terms of staining of the extracellular matrix with safranin O, there were severe reductions in 6 cases and no dye noted in 1 case. Tidemark integrity was broken in all of the individuals in this group.

The statistical data of the study were separately evaluated and calculated for Mankin scores (Table 4) and immunohistochemical results (Table 5).

4. DiscussionTunay et al. (24) showed in their studies that after the cutting of the anterior cruciate ligament (Pond–Nuki model) in rabbits, the intraarticular application of HA prevented inflammation and disintegration that formed in the cartilage tissue.

Baki (9) applied 0.1 mL of intraarticular HA 3 times at 1-week intervals to one group and the other group was injected with 0.1 mL of human amniotic fluid 3 times at 1-week intervals after medial meniscectomy in rats for

Table 3. Total Mankin scores for each subject’s tibial joint cartilage.

Rabbits BAF HA CONT

1 5 7 13

2 6 4 10

3 3 6 9

4 6 7 8

5 6 6 9

6 7 3 9

7 2 3 9

Total 35 36 67

Average 5.00 5.14 9.57

Table 4. Statistical evaluation of the Mankin scores of the femoral and tibial articular cartilages.

Bone BAF HA CONT

Femur 4.8571 ± 0.9863a

(1–9)5.0000 ± 0.7868a

(2–8)11.7143 ± 0.6801b

(9–14)

Tibia 5.0000 ± 0.6900a

(2–7)5.1429 ± 0.6700a

(3–7)11.7143 ± 0.6801b

(8–13)

a,b: Values in the same row with different letters are statistically different from each other.

280

TANRISEVER et al. / Turk J Vet Anim Sci

OA. He concluded that there was no statistically significant difference between the protective effects of HA and human amniotic fluid based on the Mankin scores at the end of the sixth week.

Tirelioğlu et al. (8) used 0.5 mL of intraarticular human amniotic fluid 3 times at 1-week intervals in the OA model that they formed after 4 weeks by cutting the anterior cruciate ligament in rabbits. They demonstrated that the damage in the medial femoral and medial tibial plateau cartilages 12 weeks after the last injection was significantly lower compared to the control group that did not receive any injections.

There are no studies in the literature using BAF in the treatment of OA. In this study, according to the Mankin scoring that was used for macroscopic and microscopic evaluation, no significant difference was found between the group treated with HA and the group treated with BAF. When both groups were compared with the control group, the Mankin scores turned out to be significantly low (P < 0.01).

Xia et al. (17) evaluated Mankin scores and immunohistochemical results of the caspase-3, caspase-8, and MMP-13 expressions with the results of millimetric wavelength irradiation at different treatment periods in rabbits with experimental OA that was created by cutting the anterior cruciate ligament. They demonstrated that the caspase-3, caspase-8, and MMP-13 staining percentages were significantly higher in the model’s control group whose cruciate ligament was cut to form OA.

In this study, caspase-3, caspase-8, and MMP-13 staining percentages were determined in order to

immunohistochemically demonstrate apoptosis, which is an indicator of OA. While no significant difference was found between the BAF and HA groups, a significant degree of difference was found between these groups and the control group with advanced OA. This significant difference clearly showed that BAF is an agent that can be used in the treatment of OA in rabbits.

In conclusion, it has been confirmed that intraarticular HA, which is widely being used in the treatment of OA in both human and veterinary practice, has shown its efficacy with this study. In histopathological and immunohistochemical examinations it has been determined that there is no significant difference between intraarticular injections of HA and BAF and that BAF does not cause any allergic reaction in the knee joints of rabbits. With these results, it has been concluded that BAF, which is the main target of this study, can be an alternative to cartilage-protecting drugs in the treatment of OA.

That being said, the therapeutic effect of BAF, which is easily obtained and more cost-efficient, can be put into practice. The investigation of its effects on other animal species and humans in the treatment of OA and the development of new treatment protocols are among the important future topics of study.

AcknowledgmentsThis study was prepared from the corresponding author’s PhD thesis and was supported financially by the Scientific and Technological Research Council of Turkey (TÜBİTAK), Project No: 113O979.

Table 5. Statistical evaluation of the immunohistochemical results of the groups.

Staining percentage BAF HA CONT P-value

Femur caspase-3 32.8571 ± 4.73804a 35.7143 ± 4.28571a 68.5714 ± 3.40068b 0.01

Tibia caspase-3 34.2857 ± 3.68856a 34.2857 ± 3.68856a 58.5714 ± 2.60820b 0.02

Femur caspase-8 31.4286 ± 2.60820a 31.4286 ± 2.60820a 71.4286 ± 2.60820b 0.01

Tibia caspase-8 31.4286 ± 1.42857a 35.7143 ± 2.97381a 70.0000 ± 3.08607b 0.01

Femur MMP-13 28.5714 ± 3.40068a 28.5714 ± 3.40068a 68.5714 ± 2.60820b 0.01

Tibia MMP-13 25.7143 ± 2.02031a 30.0000 ± 2.18218a 64.2857 ± 2.97381b 0.01

a,b: Values in the same row with different letters are statistically different from each other.

References

1. Pelletier JP, Martel-Pelletier J. Therapeutic targets in osteoarthritis: from today to tomorrow with new imaging technology. Ann Rheum Dis 2003; 62: 79-82.

2. Borrás-Verdera A, Calcedo-Bernal V, Ojeda-Levenfeld J, Clavel-Sainz C. Efficacy and safety of a single intra-articular injection of 2% hyaluronic acid plus mannitol injection in knee osteoarthritis over a 6-month period. Rev Esp Cir Ortop Traumatol 2013; 56: 274-280.

281

TANRISEVER et al. / Turk J Vet Anim Sci

3. Adams ME, Lussier AJ, Peyron JG. A risk–benefit assessment of injections of hyaluronan and its derivatives in the treatment of osteoarthritis of the knee. Drug Safety 2000; 23: 115-130.

4. Kılıç S, Karadaş E, İstek Ö, Timurkaan N. Comparison of the effectiveness of hyaluronic acid, prednisolone and doxycycline on the progression of the pond-nuki model of osteoarthritis in dogs. FÜ Sağ Bil Derg 2001; 15: 387-396.

5. Elmorsy S, Funakoshi T, Sasazawa F, Todoh M, Tadano S, Iwasaki N. Chondroprotective effects of high-molecular-weight cross-linked hyaluronic acid in a rabbit knee osteoarthritis model. Osteoarthr Cartilage 2014; 22: 121-127.

6. Dıraçoğlu D. Osteoartritte intra-artiküler hyalüronik asit tedavisi. Türk Fiz Tıp Rehab Derg 2007; 53: 154-159 (in Turkish).

7. Özgenel GY, Gülaydan F, Özcan M. Effects of human amniotic fluid on cartilage regeneration from free perichondrial grafts in rabbits. Br J Plast Surg 2004; 57: 423-428.

8. Tirelioğlu O, Bilgen MS, Atıcı T, Yalçınkaya U, Bilgen ÖF. Tavşanda deneysel osteoartrit modelinde insan amniotik sıvısının eklem içi uygulamasının kıkırdak doku ve sinovya üzerindeki etkileri. Uludağ Üniversitesi Tıp Fakültesi Dergisi 2007; 33: 121-125 (in Turkish).

9. Baki ME. Kıkırdak iyileşmesinde amniyon sıvısı ile yüksek molekül ağırlıklı hiyaluronik asitin karşılaştırılması. MSc, Karadeniz Technical University, Trabzon, Turkey, 2008 (in Turkish).

10. Dasari G, Prince I, Hearn MTW. Investigations into the rheological characteristics of bovine amniotic fluid. J Biochem Biophys Methods 1995; 30: 217-225.

11. Ravelich SR, Breier BH, Reddy S, Keelan JA, Wells DN, Pteterson AJ, Lee RSF. Insulin-like growth factor-1 and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies. Biol Reprod 2004; 70: 430-438.

12. Longaker MT, Adzick NS, Hall JL, Stair SE, Crombleholme TM, Duncan BW, Bradley SM, Harrison MR, Stern R. Studies in fetal wound healing, VII. Fetal wound healing may be modulated by hyaluronic acid stimulating activity in amniotic fluid. J Pediatr Surg 1990; 25: 430-433.

13. Decker M, Chiu ES, Dolbaum C, Moiin A, Hall J, Longaker MT, Spendlove R, Stern R. A hyaluronic acid stimulating factor from the bovine fetus and from breast cancer patients. Cancer Res 1989; 49: 3499-3505.

14. Bunning RAD, Murphy G, Kumar S, Phillips P, Reynolds JJ. Metalloproteinase inhibitors from bovine cartilage and body fluids. Eur J Biochem 1984; 139: 75-80.

15. Goggs R. Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis. Vet J 2003; 166: 140-158.

16. Kim HA, Lee YJ, Seong SC, Choe KW, Song YW.  Apoptotic chondrocyte death in human osteoarthritis. J Rheumatol 2000; 27: 455-462.

17. Xia L, Luo QL, Lin HD, Zhang JL, Guo H, He CQ. The effect of different treatment time of millimeter wave on chondrocyte apoptosis, caspase- 3, caspase-8 and MMP-13 expression in rabbit surgically induced model of knee osteoarthritis. Rheumatol Int 2012; 32: 2847-2856.

18. Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biochem Chem 1996; 271: 1544-1550.

19. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum 2001; 44: 585-594.

20. Aktaş E. Deneysel diz osteoartrit modelinde simvastatinin matriksmetalloproteinaz inhibisyonu üzerinden kondroprotektif etkisi. MSc, Gazi University, Ankara, Turkey, 1998 (in Turkish).

21. Moskovitz RW, Davis W, Sammarco J, Martens M, Baker J, Mayor M, Burstein AH, Frankel VH. Experimentally induced degenerative joint lesions following partial meniscectomy in the rabbit. Arthritis Rheum 1973; 16: 397-405.

22. Smith MM, Little CB, Rodgers K, Ghosh P. Animal models used to evaluate antiosteoarthritis drugs. Pathol Biol 1997; 45: 313-320.

23. Hayami T, Pickarski M, Zhuo Y, Wesolowski GA, Rodan GA, Duong LT. Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis. Bone 2006; 38: 234-243.

24. Tunay S, Bilgili H, Yıldız C, Yanmış I, Solakoğlu C, Gür E. Deneysel osteoartritis’in sağaltımında intraartiküler hyaluronik asit kullanımı: tavşan diz ekleminde radyolojik ve histopatolojik çalışma. Turk J Vet Anim Sci 2002; 26: 939-947 (in Turkish).

25. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg 1971; 53: 523-527.