Skip to main content
  • Research article
  • Open access
  • Published:

Effects of BIS076 in a model of osteoarthritis induced by anterior cruciate ligament transection in ovariectomised rats



Osteoarthritis (OA) is the most frequent articular disease and a leading cause of disability. There is a need for effective treatments able to slow the progression of disease. Some of the available treatments are dietary supplements providing natural components. Recent studies have shown that estrogen deficiency contributes to the pathophysiological events of OA progression.


We have used the anterior cruciate ligament transection model of OA in ovariectomised rats to study the effects of BIS076, a new formulation of a natural porcine cartilage extract associated with hydroxyapatite (as a source of calcium) and vitamin D3. Cartilage degradation, proteoglycan depletion and synovitis were followed by histochemistry. Effects on bone microstructure were determined by μCT. The levels of biomarkers in serum and inflammatory mediators in knee homogenates were measured by luminex or ELISA.


Oral administration of BIS076 reduced articular cartilage damage and serum levels of cartilage degradation markers C-telopeptide of type II collagen and cartilage oligomeric matrix protein, as well as matrix metalloproteinase-3. The local inflammatory response was down-regulated by BIS076 with lower production of pro-inflammatory cytokines and prostaglandin E2 in joint tissues. In addition, BIS076 was effective on metaphyseal bone alterations as this formulation increased volumetric bone mineral density and improved bone micro-architecture. These effects were related to the modification of bone metabolism reflected by changes in bone biomarkers with reductions in the ratio receptor activator of nuclear factor κB ligand/osteoprotegerin and the levels of tartrate-resistant acid phosphatase-5b, suggesting an inhibitory activity of BIS076 on trabecular bone resorption.


We have demonstrated the protective properties of a new formulation (BIS076) on joint lesion and bone alterations in an experimental model of OA in ovariectomised rats. This study supports the interest of BIS076 in OA treatments.

Peer Review reports


Both osteoarthritis (OA) and osteoporosis are major public health problems affecting hundreds of millions of people. Disability related to these conditions is expected to rise as a result of population aging [1,2]. OA, the most common form of rheumatic disease, is characterized by degeneration of articular cartilage, synovitis and changes to subchondral bone which exhibits altered remodelling [3,4]. The development of OA may be related to a combination of local joint-specific factors acting in the context of systemic susceptibility. Numerous systemic factors can influence OA development including genetics, age, gender, sex hormones, dietary intake and bone mineral density, as well as local mechanical factors such as muscle weakness, obesity, and joint laxity [2,5].

Osteoporosis is characterised by low bone mass and micro-architectural deterioration of bone tissue, leading to bone fragility and susceptibility to fracture [6]. Approximately 30% of all postmenopausal women have osteoporosis in the United States and Europe [7]. Age and weight are most closely associated with the development of osteoporosis although a number of other factors are involved such as height, duration of menopause and presence of chronic diseases such as hypertension, hyperlipidaemia, diabetes mellitus, and OA [8].

An inverse relationship between OA and osteoporosis has been debated over years. However, recent studies have revealed several factors which contribute to the pathogenesis of both disorders such as subchondral bone loss or inflammation [9]. Although some studies have reported a relationship between OA and increased bone mineral density [10] this feature is not associated with joint space narrowing [11]. Despite high systemic bone mineral density, bone quality and strength may be decreased at the systemic level in OA of the knee [12]. In addition, a significant decrease in periarticular subchondral bone mineral density is present in female patients with relatively mild OA of the knee [13] and structural OA changes are faster in the presence of local and systemic bone loss [14]. Given the relationship between bone resorption and cartilage damage, inhibition of bone resorption may prevent the progression of OA [15]. In fact, antiresorptive drugs have shown positive effects on bone changes and cartilage destruction in OA [16,17].

Estrogen deficiency is the cause of both the early, accelerated and the late, slow phases of bone loss in postmenopausal women and it also contributes to the continuous phase of bone loss in aging men [18]. Interestingly, epidemiologic OA studies have shown that females after the menopause are affected at a higher rate than males [5,14] and recent evidence indicates that estrogen deficiency plays a key role in the molecular pathophysiological events of joint damage and OA progression [19].

The search for treatments able to slow the progression of disease is a great challenge and would meet an important medical need for patients with OA. The rising incidence of refractory responses to available drugs has fuelled the interest in dietary supplements to provide the natural components that may help to preserve the structural integrity of joint tissues [20]. Anterior cruciate ligament transection (ACLT) in rats is a procedure that induces joint instability leading to a pathological state similar to OA. In addition, recent animal studies have shown that loss of estrogen increases damage in articular cartilage and subchondral bone [21]. In this study, we used the ACLT model of OA in ovariectomised rats to study the effects of a new formulation (BIS076) of a natural porcine cartilage extract associated with hydroxyapatite (as a source of calcium) and vitamin D3 on articular degradation and bone changes.



Female Wistar rats (9 weeks old) were obtained from Janvier (Le Genest Saint Isle, France). The animals were maintained at the animal house of the School of Pharmacy, University of Valencia, Spain, with controlled temperature (21 ± 2°C) and on a 12 h dark–light cycle. Water and food were provided ad libitum. All studies were performed in accordance with European Union regulations for the handling and use of laboratory animals. The protocols were approved by the institutional Animal Care and Use Committee (University of Valencia, Spain).

Ovariectomy and ACLT

Female Wistar rats (180–200 g body weight, 10 weeks old, n = 15 rats/group) were used in this study. Ovariectomy was performed (week 0) and 2 weeks after (week 2) ACLT was performed via an incision on the medial aspect of the right knee joint capsule. All surgical procedures were carried out under deep anaesthesia with isoflurane (1.5%) which was followed by the subcutaneous injection of butorphanol (2 mg/kg). A group of animals (Sham) underwent sham ovariectomy at week 0 followed by sham ACLT at week 2. Another group of rats (Ovx) underwent ovariectomy at week 0 followed by sham ACLT at week 2. Animals were weighed every two days throughout the experiment. There were no significant differences in body weight between groups during the study (data not shown).

Drug treatment

Rats subjected to ovariectomy + ACLT were divided into three groups: Control and two treatment groups (T1 and T2). BIS076, a preparation of a natural extract from porcine cartilage (containing 55.5% type II collagen and 21.3% glycosaminoglycans, mainly chondroitin sulfate) + hydroxyapatite + vitamin D3, was provided by Bioibérica S.A. (Barcelona, Spain). The composition of BIS076 is indicated in Table 1. BIS076 was administered daily (oral gavage of a dispersion in water) at two doses (T1: 163.5 mg/kg and T2: 245 mg/kg) from week 0 until week 12 after ovariectomy. T1 and T2 correspond approximately to daily doses in humans of 1,402 and 2,102 mg, respectively. The amount of vitamin D3 in T1 and T2 corresponds to 200 IU in humans. Sham, Ovx and ovariectomy + ACLT (Control) groups, received the same volume of vehicle (water). After week 12, animals were sacrificed. Previously, they were anesthetized with isoflurane and blood was collected by cardiac puncture. All determinations were performed at this time point.

Table 1 Components of BIS076


Knee joints were removed and fixed in 4% paraformaldehyde in phosphate-buffered saline solution. Samples were decalcified in 10% EDTA, dehydrated in graded ethanol, cleared in toluene and embedded in paraffin. Serial sections were cut (7 μm) and mounted on SuperFrost glass slides (Menzel-Gläser, Braunschweig, Germany). Sections were deparaffinised in xylol, rehydrated in graded ethanol and stained with haematoxylin and eosin (general morphology) or safranin O (proteoglycan). The sections were mounted on a DPX medium (Panreac, Barcelona, Spain) and examined under a light microscope (Nikon Eclipse E800, Izasa S.A., Valencia, Spain) with 10x objective lens and a Nikon Digital Camera DXM1200 using Nikon ACT-1 software. Histopathological grading of cartilage degradation was performed on haematoxylin-eosin and safranin O stained sections by two independent, blinded observers in accordance with the Osteoarthritis Research Society International (OARSI) histopathology grading and staging system [22]. Synovitis was scored according to Krenn et al. [23].

X-ray micro-computed tomography (μCT)

The hind paws were kept wrapped in gauze soaked in 0.9% NaCl at −20°C until analysis by μCT. Samples were analysed with a SkyScan 1172 μCT unit (Bruker microCT NV, Kontich, Belgium). Three-dimensional trabecular micro-architecture was analysed at the metaphysis and epiphysis of the tibia. Samples were imaged with an X-ray tube voltage of 50 kV, current of 200 μA, rotation step of 0.40°, at a scanning voxel size of 5.5 μm and with the use of an aluminium filter (0.5 mm in thickness). The scanning total angular rotation was 185° with an angular increment of 0.4°. Datasets were reconstructed using cone-beam reconstruction software (Skyscan NRecom) based on the Feldkamp algorithm [24] and segmented into binary images using adaptive local thresholding. Every pixel of the reconstructed 8-bit BMP images has a colour or grey value between 0 (black) and 255 (white). Morphometric indices were determined from the microtomographic datasets (integrated over a volume of interest, VOI) using direct 3D morphometry. Bone regions were obtained by free drawing regions of interest and analyzed using the commercial software provided with the equipment (SkyScan™ CT-analyzer software, version 1.7.0). For trabecular bone analysis at the metaphysis, a VOI was selected starting at a distance of 1.00 mm from the growth plate of tibial proximal metaphysis and extending a further longitudinal distance of 4.00 mm in the distal direction excluding cortical bone. For subchondral bone analysis, a VOI was selected between the subchondral plate and the growth plate of the proximal metaphysis. Thresholding was applied to the images to segment the trabecular bone from the background and the same threshold setting was used for all the samples. Morphometric indices were determined from the microtomographic data sets (integrated over a VOI) using direct 3D morphometry. Total volume of VOI (tissue volume; TV; mm3) and trabecular bone volume (BV; mm3) were calculated based on the hexahedral marching cubes volume model of the VOI. Trabecular bone volume (BV/TV; %) was directly calculated. Trabecular thickness (Tb.Th; mm), trabecular separation (TbS; mm) and trabecular number (Tb.N; 1/mm) were measured directly on 3D images. Measurements of Tb.Th were calibrated by scanning and analyzing three aluminum foils with thicknesses of 50, 125 and 250 μm. The non-metric indices, such as trabecular bone pattern factor (Tb.Pf; 1/mm) were also calculated using the direct 3D model.

Determination of inflammatory mediators

The knees were amputated, skin was dissected and the rest of tissue was immediately frozen in liquid nitrogen and pulverized in a Freezer/Mill 6750 (Spex SamplePrep, Metuchen, NJ, USA). The powder was extracted with 2 ml of A buffer pH 7.4 [10 mM 4-(hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES, pH 8), 1 mM EDTA, 1 mM ethylene glycol bis(β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM KCl, 1 mM dithiothreitol, 5 mM NaF, 1 mM Na3VO4, 1 μg/ml leupeptin, 0.1 μg/ml aprotinin and 0.5 mM phenylmethylsulfonyl fluoride]. After the first centrifugation at 1,200 g (10 min at 4°C), supernatants were collected, centrifuged at 9,000 g (15 min at 4°C) and used to measure cytokines by ELISA. Interleukin(IL)-1β and tumour necrosis factor-α (TNFα) were measured by using the kits from R&D Systems (Minneapolis, MN, USA) with a sensitivity of 5.0 pg/ml, and IL-6 and IL-17A with the kits from eBioscience (San Diego, CA, USA) with a sensitivity of 12.0 and 1.0 pg/ml, respectively. Prostaglandin E2 (PGE2) was determined by radioimmunoassay [25].

Determination of serum biomarkers

Serum was used for the determination by ELISA of C-telopeptide of type II collagen (CTX-II, serum pre-clinical Cartilaps, Nordic Biosciences, Herlev, Denmark, sensitivity of 6.3 pg/ml), cartilage oligomeric matrix protein (COMP, MD Biosciences, Zürich, Switzerland, sensitivity <0.2 U/l), matrix metalloproteinase (MMP)-3 (R&D Systems, sensitivity of 19.0 pg/ml), N-terminal propeptide of type I procollagen (PINP, IDS Inc., Boldon, UK, sensitivity of 0.7 ng/ml) and tartrate-resistant acid phosphatase (TRAP)-5b (sensitivity of 0.1 U/l). Serum levels of osteocalcin (OC), osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) were determined by luminex, with sensitivity of 7.0, 2.3 and 3.3 pg/ml, respectively (Millipore Corporation, Billerica, MA, USA). Alkaline phosphatase (ALP) levels in serum were determined as previously described [26].

Statistical analysis

The results are presented as mean ± standard deviation (SD), n: number of animals. The level of statistical significance was determined by using one-way analysis of variance (ANOVA) followed by Bonferroni’s test. Scoring was analyzed by a non-parametric test (Kruskal-Wallis followed by Dunn's post-test).


Effect of BIS076 on histopathological changes in the joint

The histopathological analysis showed the presence of cartilage damage in Control rats (Figures 1 and 2). Structural changes in cartilage surface included focal damage and fibrillation, loss of proteoglycan, decreased chondrocyte cell number and increased chondrocyte clustering. These pathological changes were reflected by the score of the joint in Control group which was significantly higher than the sham group (Figure 3). The grade of cartilage damage in the groups treated with BIS076 (T1 and T2) was significantly lower than in Control group and not statistically different from the sham-operated group. In addition, Control rats showed synovitis which was scored according to Krenn et al. [23]. In rats treated with BIS076 at both doses (T1 and T2), a lower level of synovitis was observed compared with Control animals although it did not reach statistical significance.

Figure 1
figure 1

Histochemical analysis in the ACLT knees. Representative images of sections from femur, tibia, patella stained with haematoxylin and eosin. Bars: 200 μm and 100 μm.

Figure 2
figure 2

Histochemical analysis in the ACLT knees. Representative images of sections from femur, tibia, patella stained with safranin O. Bars: 200 μm and 100 μm.

Figure 3
figure 3

Cartilage degradation and synovitis were scored in frontal sections of knee joints as indicated in materials and methods. Cartilage degradation was determined in accordance with the Osteoarthritis Research Society International (OARSI) histopathology grading and staging system [22]. Synovitis was scored according to Krenn et al. [23]. Results are expressed as mean ± SD (n = 5); ++P < 0.01 with respect to Sham; *P < 0.05 compared to Control (Kruskal-Wallis followed by Dunn's post-test).

Effect of BIS076 on bone micro-architecture

μCT analysis indicated loss of bone mass and structural alterations in Ovx and Control rats compared with Sham animals (Table 2 and Figure 4). Main bone changes were observed in metaphysis, with significant reductions in bone volume fraction (BV/TV), bone surface density (BS/TV), trabecular number (Tb.N) and volumetric bone mineral density (vBMD) whereas trabecular factor (Tb.Pf) significantly increased. Similar changes were observed in the parameters measured in epiphysis (Ovx or Control versus Sham) although they did not reach statistical significance. Interestingly, BIS076 counteracted all bone alterations shown in Control animals in the metaphysis only, with statistically significant effects at the higher dose (T2).

Table 2 Effect of BIS076 on bone parameters after μCT analysis
Figure 4
figure 4

Representative images of epiphysis and metaphysis of tibia by μCT analysis. Quantification of trabecular bone parameters in both regions is shown in Table 2.

Effect of BIS076 on inflammatory mediators in the joint

We have shown previously the contribution of inflammation to the progression of OA after ACLT [27]. The levels of the inflammatory mediators IL-1β, IL-6, IL-17, TNFα and PGE2 were determined in knee homogenates. Figure 5 shows that IL-1β, IL-17 and PGE2 were significantly higher in the Control compared with the Sham group. Treatment with BIS076 reduced the expression of inflammatory mediators with significant effects for T1 and T2 on cytokines IL-1β, IL-6 and IL-17, and for T2 on PGE2.

Figure 5
figure 5

Levels of pro-inflammatory mediators in homogenates from ACLT knees. IL-1β, IL-6, IL-17 and TNFα were measured by ELISA. PGE2 was measured by radioimmunoassay. Results show mean ± SD (n = 7); +P < 0.05 with respect to Sham; *P < 0.05, **P < 0.01 compared to Control (ANOVA followed by Bonferroni’s test).

Effect of BIS076 on serum biomarkers

The cartilage degradation markers CTX-II and COMP were measured in serum. Control animals showed enhanced levels of these molecules with respect to sham-operated rats (Figure 6). BIS076 reduced the expression of cartilage degradation markers with a significant effect at the higher dose (T2). MMP-3, an inflammatory marker related with cartilage destruction in OA [28] was also assessed. The levels of MMP-3 observed in Control rats were significantly reduced by BIS076 treatment at the higher dose (T2). The levels of bone turnover markers were also determined. The levels of ALP significantly increased and those of TRAP5b decreased in the groups Ovx and Control. BIS076 at both doses (T1 and T2) significantly reduced the increase of ALP activity shown in Control rats, as well as TRAP5b levels and the RANKL/OPG ratio whereas OC was significantly enhanced by the higher dose (T2) of BIS076.

Figure 6
figure 6

Levels of serum biomarkers. COMP, CTX-II, MMP-3, PINP and TRAP-5b were measured by ELISA. Serum levels of OC, OPG and RANKL were determined by luminex. ALP levels in serum were determined as previously described [26]. Results show mean ± SD (n = 12-15); +P < 0.05, ++P < 0.01 with respect to Sham; *P < 0.05, **P < 0.01compared to Control (ANOVA followed by Bonferroni’s test).


Disruption of the balance between degradative and repair processes leads to loss of cartilage integrity in OA. Cartilage degeneration follows joint instability after ACLT and it is associated with localized destruction of the type II collagen network [29]. In this study, OA was induced by ACLT in ovariectomised rats. This experimental model allows the evaluation of treatments able to act on joint degradation and bone alterations related to OA and osteoporosis. We analyzed the effects of BIS076, a new formulation of a natural porcine cartilage extract associated with hydroxyapatite and vitamin D3 and found that important alterations in cartilage, synovial membrane and bone induced in Control rats were improved in animals treated with BIS076.

Radiographic severity of knee OA is associated with serum or urinary levels of cartilage turnover markers such as CTX-II and COMP [30,31]. In this experimental model, these biomarkers were increased in the serum of Control rats with respect to sham-operated animals at the end of the experiment. The protection exerted by BIS076 against cartilage degradation was reflected by the reduction in the levels of CTX-II and COMP. In addition, BIS076 down-regulated MMP-3, an inflammatory marker that may be a predictor of radiographic OA progression [28].

Cartilage homeostasis is maintained at the normal joint by a balance between anabolic and catabolic processes. Nevertheless, catabolic events leading to cartilage destruction predominate in OA. Overloading and inflammation are factors contributing to knee OA. It is accepted that inflammatory changes mediate the progression of joint lesion [32,33] and activation of chondrocytes by mechanical stress results in the production of inflammatory mediators. In fact, synovitis and inflammatory mediators such as cytokines and chemokines are present in early-stage and last-stage disease. Pro-inflammatory cytokines induce catabolic enzymes and cartilage matrix degradation [34] leading to an imbalance between cartilage matrix degradation and repair. In turn, cartilage matrix molecules and degradation products can induce inflammatory responses creating a vicious circle [35]. In addition, pro-inflammatory cytokines induce cyclooxygenase-2 and microsomal PGE synthase-1 in articular tissues leading to an enhanced production of PGE2 that may contribute to synovitis and cartilage degradation [36,37]. Our data indicate that BIS076 lowers the production of pro-inflammatory cytokines and PGE2 in the affected joint. Although further studies would be necessary to determine the mechanism of action of this formulation, it is likely that its anti-inflammatory effects contribute to the reduction in OA progression.

On the other hand, trabecular bone alterations in metaphysis were improved by BIS076, with increases in volumetric bone mineral density, bone volume fraction, bone surface density, trabecular number and connectivity. These effects were related to the modification of bone metabolism by BIS076 which partly counteracted the increase in ALP activity shown in Control rats and enhanced OC levels at the highest dose. Interestingly, this formulation reduced the RANKL/OPG ratio, an index of osteoclastic activation [38,39], and the levels of the resorption marker TRAP5b, suggesting that the protective effects of BIS076 are related to a decreased trabecular bone resorption.

The different components of BIS076 may contribute to the observed effects. In addition to the well-known role of vitamin D3 and calcium in bone metabolism, some studies have suggested a protective effect of this vitamin on OA [40]. Nevertheless, clinical trials of vitamin D supplementation have failed to show any reduction in knee pain or cartilage volume loss in patients with symptomatic knee OA [41]. Therefore, it is likely that collagen and glycosaminoglycan components of BIS076 play a main role in experimental OA. It is interesting to note that hydrolysed collagen enhances the synthesis of type II collagen by bovine chondrocytes [42] and clinical studies have suggested that oral ingestion of this product reduces pain in OA patients and it may have an additive effect with calcitonin on osteoporosis [43]. Furthermore, previous reports have suggested that chondroitin sulfate exerts some anabolic effects on cartilage and also inhibits nuclear factor-κB activation leading to the down-regulation of inflammatory mediators and catabolic enzymes such as MMPs thus preventing cartilage degradation [44].


This study has demonstrated that BIS076 significantly reduces the severity of structural changes in joint tissues, down-regulates the inflammatory response and improves metaphyseal bone density and micro-architecture in a model of OA induced by ACLT in ovariectomised rats. These findings suggest that BIS076 is a new promising and effective formulation for OA and could have a potential role in bone protection during treatment.



Anterior cruciate ligament transection


Alkaline phosphatase


Analysis of variance


Cartilage oligomeric matrix protein


C-Telopeptide of type II collagen




Matrix metalloproteinase




Osteoarthritis research society international






N-terminal propeptide of type I procollagen


Prostaglandin E2


Receptor activator of nuclear factor κB ligand


Tumor necrosis factor-α


Tartrate-resistant acid phosphatase.


  1. Strom O, Borgstrom F, Kanis JA, Compston J, Cooper C, McCloskey EV, et al. Osteoporosis: burden, health care provision and opportunities in the EU: a report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA). Arch Osteoporos. 2011;6(1–2):59–155.

    Article  CAS  PubMed  Google Scholar 

  2. Suri P, Morgenroth DC, Hunter DJ. Epidemiology of osteoarthritis and associated comorbidities. PM R. 2012;4(5 Suppl):S10–9.

    Article  PubMed  Google Scholar 

  3. Radin EL, Rose RM. Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop Relat Res. 1986;213(12):34–40.

    PubMed  Google Scholar 

  4. Hopwood B, Tsykin A, Findlay DM, Fazzalari NL. Microarray gene expression profiling of osteoarthritic bone suggests altered bone remodelling, WNT and transforming growth factor-beta/bone morphogenic protein signalling. Arthritis Res Ther. 2007;9(5):R100.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, et al. Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med. 2000;133(8):635–46.

    Article  CAS  PubMed  Google Scholar 

  6. Consensus Development Conference. Diagnosis, prophylaxis and treatment of osteoporosis. Am J Med. 1993;94(6):646–50.

    Article  Google Scholar 

  7. Reginster JY, Burlet N. Osteoporosis: a still increasing prevalence. Bone. 2006;38(2 Suppl 1):S4–9.

    Article  PubMed  Google Scholar 

  8. Kim SK, Yoo TK, Oh E, Kim DW. Osteoporosis risk prediction using machine learning and conventional methods. Conf Proc IEEE Eng Med Biol Soc. 2013;2013:188–91.

    PubMed  Google Scholar 

  9. Bultink IE, Lems WF. Osteoarthritis and osteoporosis: what is the overlap? Curr Rheumatol Rep. 2013;15(5):328.

    Article  PubMed  Google Scholar 

  10. Foss MV, Byers PD. Bone density, osteoarthrosis of the hip, and fracture of the upper end of the femur. Ann Rheum Dis. 1972;31(4):259–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hannan MT, Anderson JJ, Zhang Y, Levy D, Felson DT. Bone mineral density and knee osteoarthritis in elderly men and women. The Framingham Study. Arthritis Rheum. 1993;36(12):1671–80.

    Article  CAS  PubMed  Google Scholar 

  12. Lee S, Kim TN, Kim SH. Knee osteoarthritis is associated with increased prevalence of vertebral fractures despite high systemic bone mineral density: a cross-sectional study in an Asian population. Mod Rheumatol. 2014;24(1):172–81.

    Article  PubMed  Google Scholar 

  13. Karvonen RL, Miller PR, Nelson DA, Granda JL, Fernandez-Madrid F. Periarticular osteoporosis in osteoarthritis of the knee. J Rheumatol. 1998;25(11):2187–94.

    CAS  PubMed  Google Scholar 

  14. Arden N, Nevitt MC. Osteoarthritis: Epidemiology. Best Pract Res Clin Rheumatol. 2006;20(1):3–25.

    Article  PubMed  Google Scholar 

  15. Kadri A, Funck-Brentano T, Lin H, Ea HK, Hannouche D, Marty C, et al. Inhibition of bone resorption blunts osteoarthritis in mice with high bone remodelling. Ann Rheum Dis. 2010;69(8):1533–8.

    Article  PubMed  Google Scholar 

  16. Panahifar A, Maksymowych WP, Doschak MR. Potential mechanism of alendronate inhibition of osteophyte formation in the rat model of post-traumatic osteoarthritis: evaluation of elemental strontium as a molecular tracer of bone formation. Osteoarthritis Cartilage. 2012;20(7):694–702.

    Article  CAS  PubMed  Google Scholar 

  17. Moreau M, Rialland P, Pelletier JP, Martel-Pelletier J, Lajeunesse D, Boileau C, et al. Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model. Arthritis Res Ther. 2011;13(3):R98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Riggs BL, Khosla S, Melton III LJ. A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res. 1998;13(5):763–73.

    Article  CAS  PubMed  Google Scholar 

  19. Herrero-Beaumont G, Roman-Blas JA, Castaneda S, Jimenez SA. Primary osteoarthritis no longer primary: three subsets with distinct etiological, clinical, and therapeutic characteristics. Semin Arthritis Rheum. 2009;39(2):71–80.

    Article  PubMed  Google Scholar 

  20. Vista ES, Lau CS. What about supplements for osteoarthritis? a critical and evidenced-based review. Int J Rheum Dis. 2011;14(2):152–8.

    Article  PubMed  Google Scholar 

  21. Sniekers YH, Weinans H, van Osch GJ, Van Leeuwen JP. Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis. Arthritis Res Ther. 2010;12(5):R182.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage. 2006;14(1):13–29.

    Article  CAS  PubMed  Google Scholar 

  23. Krenn V, Morawietz L, Burmester GR, Kinne RW, Mueller-Ladner U, Muller B, et al. Synovitis score: discrimination between chronic low-grade and high-grade synovitis. Histopathology. 2006;49(4):358–64.

    Article  CAS  PubMed  Google Scholar 

  24. Feldkamp LA, Davis LC, Krettek C. Practical cone-beam algorithm. J Opt Soc Am A. 1984;1(1):612–9.

    Article  Google Scholar 

  25. Moroney MA, Alcaraz MJ, Forder RA, Carey F, Hoult JRS. Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids. J Pharm Pharmacol. 1988;40(11):787–92.

    Article  CAS  PubMed  Google Scholar 

  26. Kind PR, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. J Clin Pathol. 1954;7(4):322–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Braza-Boils A, Alcaraz MJ, Ferrandiz ML. Regulation of the inflammatory response by tin protoporphyrin IX in the rat anterior cruciate ligament transection model of osteoarthritis. J Orthop Res. 2011;29(9):1375–82.

    Article  CAS  PubMed  Google Scholar 

  28. Lohmander LS, Brandt KD, Mazzuca SA, Katz BP, Larsson S, Struglics A, et al. Use of the plasma stromelysin (matrix metalloproteinase 3) concentration to predict joint space narrowing in knee osteoarthritis. Arthritis Rheum. 2005;52(10):3160–7.

    Article  CAS  PubMed  Google Scholar 

  29. Stoop R, Buma P, van der Kraan PM, Hollander AP, Billinghurst RC, Meijers TH, et al. Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats. Osteoarthritis Cartilage. 2001;9(4):308–15.

    Article  CAS  PubMed  Google Scholar 

  30. Garnero P, Piperno M, Gineyts E, Christgau S, Delmas PD, Vignon E. Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage. Ann Rheum Dis. 2001;60(60):619–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Clark AG, Jordan JM, Vilim V, Renner JB, Dragomir AD, Luta G, et al. Serum cartilage oligomeric matrix protein reflects osteoarthritis presence and severity: the Johnston County Osteoarthritis Project. Arthritis Rheum. 1999;42(11):2356–64.

    Article  CAS  PubMed  Google Scholar 

  32. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2003;39(1–2):237–46.

    Google Scholar 

  33. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum. 2001;44(6):1237–47.

    Article  CAS  PubMed  Google Scholar 

  34. Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res. 2004;427(Suppl):S27–36.

    Article  PubMed  Google Scholar 

  35. Sellam J, Berenbaum F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis: Nat Rev. Rheumatol. 2010;6(11):625–35.

    CAS  Google Scholar 

  36. Hardy MM, Seibert K, Manning PT, Currie MG, Woerner BM, Edwards D, et al. Cyclooxygenase 2-dependent prostaglandin E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants. Arthritis Rheum. 2002;46(7):1789–803.

    Article  CAS  PubMed  Google Scholar 

  37. Attur M, Al Mussawir HE, Patel J, Kitay A, Dave M, Palmer G, et al. Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor. J Immunol. 2008;181(7):5082–8.

    Article  CAS  PubMed  Google Scholar 

  38. Jabbar S, Drury J, Fordham JN, Datta HK, Francis RM, Tuck SP. Osteoprotegerin, RANKL and bone turnover in postmenopausal osteoporosis. J Clin Pathol. 2011;64(4):354–7.

    Article  CAS  PubMed  Google Scholar 

  39. Ibañez L, Ferrandiz ML, Brines R, Guede D, Cuadrado A, Alcaraz MJ. Effects of Nrf2 deficiency on bone microarchitecture in an experimental model of osteoporosis. Oxid Med Cell Longev. 2014;2014:726590.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Heidari B, Heidari P, Hajian-Tilaki K. Association between serum vitamin D deficiency and knee osteoarthritis. Int Orthop. 2011;35(11):1627–31.

    Article  PubMed  Google Scholar 

  41. McAlindon T, Lavalley M, Schneider E, Nuite M, Lee JY, Price LL, et al. Effect of vitamin D supplementation on progression of knee pain and cartilage volume loss in patients with symptomatic osteoarthritis: a randomized controlled trial. JAMA. 2013;309(2):155–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Oesser S, Seifert J. Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen. Cell Tissue Res. 2003;311(3):393–9.

    CAS  PubMed  Google Scholar 

  43. Moskowitz RW. Role of collagen hydrolysate in bone and joint disease. Semin Arthritis Rheum. 2000;30(2):87–99.

    Article  CAS  PubMed  Google Scholar 

  44. du Souich P. Absorption, distribution and mechanism of action of SYSADOAS. Pharmacol Ther. 2014;142(3):362–74.

    Article  PubMed  Google Scholar 

Download references


The present study was supported by grants from Bioibérica S.A. (348/2010) and Spanish Ministerio de Economía y Competitividad, ISCIII, FEDER (RETICEF RD12/0043/0013). The funding sources had no role in the conduct of the study; collection, management, analysis, interpretation of the data; and preparation of the manuscript. The authors thank Ms. Anna Blanco for her technical assistance.

Author information

Authors and Affiliations


Corresponding author

Correspondence to María José Alcaraz.

Additional information

Competing interests

A.T., P.D., R.R., E.M. and J.V. are employed by Bioibérica S.A.

Authors’ contributions

A.T., P.D., R.R., E.M, J.V., M.L.F., M.J.A. conceived and designed the experiments. M.L.F., M.C.T., M.C.C. performed the experiments. M.L.F., M.C.T., M.J.A. analysed the data and wrote the paper. All authors read and approved the final manuscript.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ferrándiz, M.L., Terencio, M.C., Carceller, M.C. et al. Effects of BIS076 in a model of osteoarthritis induced by anterior cruciate ligament transection in ovariectomised rats. BMC Musculoskelet Disord 16, 92 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: