Connective Tissue Research, 2013; 54(2): 139–146Copyright © Informa Healthcare USA, Inc.ISSN: 0300-8207 print/1607-8438 onlineDOI: 10.3109/03008207.2012.760549

The Potential Protective Effects of Calcitonin Involved in CoordinatingChondrocyte Response, Extracellular Matrix, and Subchondral TrabecularBone in Experimental Osteoarthritis

Tan Cheng,1 Liu Zhang,1,2 Xiaoxia Fu,3 Wenya Wang,3 Hong Xu,1 Huiping Song,2 Yingze Zhang4

1Department of Orthopedic Surgery, Hebei Medical University, Shijiazhuang, China, 2Department of Orthopedic Surgery, Affiliated Hospital ofHebei United University, Tangshan, China, 3Department of Pathology, School of Basic Medical Sciences, Hebei United University, Tangshan,China, 4Department of Orthopedic Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, China

Previous reports indicate a potential role for calcitonin (CT) in thetreatment of osteoarthritis (OA). To evaluate this potential ther-apeutic role, we investigated the effect of CT pretreatment on theactivation of mitogen-activated protein kinase (MAPK) signalingand the expression of matrix metalloproteinase-13 (MMP-13) ininterleukin-1β (IL-1β)-induced chondrocytes, and further assessedits protective effect in a rat model of anterior cruciate ligamenttransection (ACLT), using sham-operated and saline-treated con-trols. Using western blotting in vitro, we found that CT pretreat-ment inhibited the IL-1β-induced phosphorylation of 38,000-dalton protein (p38) and extracellular regulated protein 1/2(ERK1/2) and reduced the expression of MMP-13 protein. For thein vivo experiment, 30 male rats were randomly divided into threegroups of 10, subjected to bilateral ACLT or sham surgery, andthen treated for 12 weeks with subcutaneous injections of CT ornormal saline. Histological observations showed that CT treat-ment reduced the severity of the cartilage lesions stemmingfrom the ACLT surgery and provided a lower Mankin score whencompared with that determined for rats in the saline-treated ACLTgroup. Immunohistochemical staining revealed that CT treatmentincreased type II collagen expression and decreased MMP-3 and adisintegrin and metalloproteinase with thrombospondin motifs-4(ADAMTS-4) expression when compared with the saline-treatedgroup. Subchondral bone analysis indicated that CT treatmentinhibited the reduction in bone mineral density observed in thesaline-treated ACLT group and reduced the ACLT-induceddestruction to the subchondral trabecular microstructure. Ourdata demonstrate that CT induces its protective effects by redu-cing the chondrocyte response to inflammatory stimuli, cartilageextracellular matrix degradation, and subchondral trabecularmicrostructure damages brought on by OA.

Keywords: osteoarthritis, calcitonin, chondrocyte, extracellularmatrix, subchondral trabecular bone

Introduction

Osteoarthritis (OA) is one of the most common joint diseasesworldwide, posing a serious public health concern as the popula-tion continues to age [1]. The disease results in diarthrosis dys-function in humans and a reduced quality of life for these patients.The most common risk factors for the development of OA are anincrease in inflammatory factors and abnormal biomechanics [2],

both of which are believed to trigger articular cartilage degradationand subchondral bone turnover. The current treatment strategiesfor OA are aimed at alleviating the symptoms associated with thedisease, but fall short of being able to suppress the destructiveeffects of OA in these patients [1]. Therefore, new therapeuticstrategies are aimed at identifying a safe and effective disease-modifying drug for OA patients [3].

In experimental models of OA, several treatments have beenreported to provide beneficial effects to the joints, which haveincluded the use of glucosamine, matrix metalloproteinase(MMP) inhibitors, bisphosphonates, and cytokine inhibitors [4].Recently, the use of calcitonin (CT) has been discussed as an idealtreatment against the progression of OA. Preliminary results haveshown that CT treatment has a protective effect on articularcartilage and subchondral bone in models of OA [5]. It is knownthat CT not only has a physiological role in regulating calciumhomeostasis [6] but also binds to CT receptors on osteoclasts tomediate bone metabolism. In the clinic, the hormone has beenused to treat osteoporosis for more than three decades. Hence, thesafety profile of CT has been recognized for the treatment ofpatients with osteoporosis.

Intriguingly, a new pharmacological characteristic of CT sug-gests its potential therapeutic benefit in the treatment of OA. Aprevious experimental study revealed that CT reduced the biochem-ical markers of cartilage degradation [C-propeptide of type II col-lagen (CTX-II)] [7]. Moreover, CT receptors in human OAchondrocytes have been identified in vitro [8]. These results may,in part, explain the potential chondroprotective effect of CT. Inaddition, CT treatment improved the subchondral bone volume ina canine model of OA, which is considered to be the safeguard forarticular cartilage [9], as changes to both the articular cartilage andthe subchondral bone lead to the pathological progression of OA[10,11].

Chondrocytes and extracellular matrix are the key struc-tural components of cartilage. Chondrocytes synthesize andsecrete collagen and aggrecan, the major backbone compo-nents of the extracellular matrix network, which function toabsorb the impact from joint loading. Macroscopically, abreakdown in the extracellular matrix eventually presents asarticular cartilage degeneration and, in the initial stage of OA,abnormal biomechanics impacts upon subchondral boneremodeling, resulting in an accumulation of trabecular

Address correspondence to Liu Zhang, MD, PhD, Department of Orthopedic Surgery, Hebei Medical University, Shijiazhuang, China. E-mail: zhliu130@sohu.comReceived 20 August 2012; Revised 17 December 2012; Accepted 17 December 2012

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micro-damage, which in turn accelerates the progression ofOA.

In another study, using a canine experimental OA model, CTwas found to reduce the serum levels of antigenic keratan sulfate,a biochemical marker of cartilage, and hyaluronan, a biochemicalmarker of synovial fluid, as well as reduce the urinary levels ofdeoxy-pyridinoline and pyridinoline, both biochemical markers ofbone [12]. A recent controlled and randomized trial showed thatoral CT in combination with a carrier molecule can also inhibit theexpression ofMMP-mediated cartilage biomarkers and osteoclast-mediated bone biomarkers [13]. This evidence has helped tounveil the dual effects of CT on cartilage and bone metabolismin OA treatment [7,12]. However, the potential protective effect ofCT in OA remains to be tested.

In this study, we tested the potential chondroprotective effectsof CT on chondrocyte response to interleukin-1β (IL-1β)-inducedmetalloproteinase-13 (MMP-13) expression via mitogen-activated protein kinase (MAPK) signaling in vitro and examinedits effect on the extracellular matrix integrity by assessing type IIcollagen, MMP-3, and a disintegrin and metalloproteinase withthrombospondin motifs-4 (ADAMTS-4) protein expressionsin vivo. We also quantitatively analyzed the effects of CT onboth the subchondral bone mineral density (BMD) and the sub-chondral trabecular microstructure using histomorphometry. Thecurrent knowledge from this study is expected to further clarify theeffect of CT on the entire joint during the progression of OA,including its effect on chondrocytes, the extracellular matrix, andthe subchondral trabecular bone.

Materials and Methods

Cell CultureChondrocytes from rat articular cartilage of the knee joint werecultured in Dulbecco’s modified eagle’s medium with 10% fetalbovine serum at 37�C with 5% CO2. Chondrocytes in their secondpassage were used for subsequent experiments. Chondrocyte cul-tures were divided into six groups as follows: no treatment (con-trol group), induction with IL-1β (10 ng/mL) for 15 min (IL-1βgroup), pretreatment with low-dose (50 ng/mL; L group) or high-dose (500 ng/mL; H group) of CT for 24 hr, followed by IL-1βinduction (L þ IL-1β group or H þ IL-1β group), and treatmentwith low- or high-dose CT for 24 hr in the absence of IL-1βinduction (L group or H group).

Western Blot Analysis of p38, ERK1/2, JNK, and MMP-13Chondrocytes were lysed using radio immunoprecipitation assay(ZO2338A; Aidlab Biotechnologies Co. Ltd., Beijing, China), andthe total protein content was quantified by a protein assay(PC0020; Solarbio Science and Technology Co. Ltd., Beijing,China). The proteins were fractionated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred tonitrocellulose membranes. Membranes were blocked with 5%nonfat milk and incubated overnight at 4�C with one of thefollowing primary antibodies: mouse polyclonal antibodies againsttotal 38,000-dalton protein (p38) (sc-136210), phospho-p38 (sc-7973), total extracellular regulated protein 1/2 (ERK1/2) (sc-135900), phospho-ERK1/2 (sc-81492), total c-Jun N-terminalKinase (JNK) (sc-7345), phospho-JNK (sc-6254) (all from SantaCruz Biotechnology Inc., Santa Cruz, CA, USA), or rabbit anti-MMP-13 antibody (BA0574; Boster Bio-engineering Co., Ltd.,Wuhan, China). Membranes were then washed and incubatedwith the appropriate alkaline phosphatase-conjugated secondaryantibody (Boster Bio-engineering Co., Ltd., Wuhan, China) for

1 hr at 4�C. The protein bands were visualized following incuba-tion with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt/nitroblue tetrazolium chloride. The results were normalizedagainst rabbit antiglyceraldehyde-3-phosphate (GAPDH) levels(anti-GAPDH; BA2913; Boster Bio-engineering Co., Ltd,Wuhan, China). The protein expressions were analyzed by inte-grated optical density (IOD), using Image-Pro Plus version 6.0software (Media Cybernetics Inc., Bethesda, FL, USA).

Animal HandlingAll experiments were approved by Hebei United UniversityAnimal Care and Use Committee. Three-week-old, maleSprague-Dawley rats, weighing 200 � 15 g (Vital RiverExperimental Animal Technical Co., Ltd, Beijing, China) werefed a standard rodent diet and housed in the Center ofExperimental Care. Thirty rats were randomly divided into threegroups (n ¼ 10 rats per group). The rats were subjected to shamoperation in the control group (Sham group), anterior cruciateligament transection (ACLT) surgery supplemented with normalsaline (NS) (ACLT þ NS group), or ACLT surgery supplementedwith CT (ACLT þ CT group). ACLT surgery was performedaccording to the previously described protocols [14].Immediately following the surgery, rats were subjected to differenttreatment regimens for 12 weeks postsurgery as follows: in theACLT þ CT group, rats were subcutaneously injected with CT(5 U/kg) every 2 days, and in the ACLTþ NS and control groups,rats received 0.9% normal saline treatment. All rats were labeledwith calcein (10 mg/kg) and tetracycline (10 mg/kg) and injectedsubcutaneously at 11 and 4 days prior to sacrifice, respectively.

Cartilage Gross Morphological Observation and SubchondralBMD AssessmentThe gross appearance of the distal femur and the proximal tibiawas recorded by a digital camera (Canon 550D, Tokyo, Japan).BMD of the medial and lateral tibial plateaus was detected by dualX-ray absorptiometry (DXA, Norland RX, USA). The region ofinterest (ROI) was a 3 mm � 4 mm area from the subchondralbone plate to the epiphyseal plate. BMD within the ROI wasmeasured by a small animal model resolution software program.Measurements from 10 knees per group were averaged.

Tissue Preparation and Cartilage Histopathological AnalysisAll femurs were fixed in 70% ethanol for 48 hr and decalcified with15% ethylene diamine tetraacetic acid disodium (EDTA-Na2) (pH7.4) at 4�C for 6 weeks. Tissues were dehydrated, embedded inparaffin, and cut into 5-μm-thick sections, according to standardprotocols. Four sections from each sample were stained withSafranin-O/fast green and hematoxylin-eosin (HE), respectively.Then, three-color digital images from each section were recordedby light microscopy (Olympus BX61, Olympus, Japan).Semiquantitative histopathological analysis was establishedaccording to the Mankin score system [15] using four character-istics: structure, cellularity, matrix staining, and tidemarkintegrity.

Cartilage Immunohistochemistry for Type II Collagen,ADAMTS-4, and MMP-3Each section was deparaffinized in xylene, rehydrated in gradedalcohol, washed with PBS (pH 7.4) for 6 min, immersed in 3%hydrogen peroxide for 10 min, and repaired with complex digestenzyme for 40 min. Polyclonal primary antibodies for immuno-histochemistry were purchased from Boster Bio-engineeringCompany (Wuhan, China): rabbit anti-type II collagen

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(BA0533), rabbit anti-ADAMTS-4 (BA3375), and rabbit anti-MMP-3 (BA1272) antibodies. All antibodies were diluted 1:100and incubated with tissues overnight at 4�C. Each section waswashed three times in PBS (pH 7.4). Sections were further incu-bated with horseradish peroxidase-conjugated secondary antibo-dies for 60 min at 37�C. Colorimetric detection with 3,30-diaminobenzidine substrate (Zhongshan GoldenbridgeBiotechnology Co., Ltd., Beijing, China) was performed for allsections subsequent to counterstaining the nuclei with hematox-ylin. Protein expression was quantified by IOD, using IPP version6.0 software (Media Cybernetics Inc., Silver Spring, MD, USA).

Subchondral Histomorphometric MeasurementsUndecalcified samples from the proximal end of each tibia wereembedded in methyl methacrylate, cut into 8-μm-thick sections,and stained with Giemsa stain. Structural parameters of the sub-chondral bone were measured by light microscope (OlympusBX61, Olympus, Japan), including bone volume (BV/TV), trabe-cular thickness (Tb.Th), trabecular number (Tb.N), and trabecularseparation (Tb.Sp). Dynamic parameters of the subchondral bone,including mineralizing surface (MS/BS), mineral apposition rate(MAR), and bone formation rate (BFR/BS), were computed usingconfocal laser scanning microscopy (FLUOVIEW FV1000,Olympus, Japan). Histomorphometrical parameters were assessedusing IPP version 6.0 software (Media Cybernetics Inc.) accordingto the guidelines of the American Society of Bone and MineralResearch [16]. Measurements from 10 right knees per group wereaveraged.

Statistical AnalysisAll data are expressed as the mean� standard deviation. Multiplegroup comparisons were conducted using analysis of variance.Group differences resulting in p-values of less than 0.05 wereconsidered to be statistically significant.

Results

Roles of MAPK and MMP-13 in the Chondroprotective Effectsof CTTo investigate the in vitro effects of CT on chondrocytes, westimulated chondrocytes with IL-1β with or without pretreatmentwith two concentrations of CT. Stimulation of rat chondrocytes byIL-1β induced the phosphorylation of p38, ERK, and JNK(Figure 1), as well as MMP-13, when compared with unstimulatedcontrols. Importantly, pretreatment with CT (50 and 500 ng/mL)differentially inhibited the phosphorylation levels of p38(68.59 � 0.88 and 67.42 � 0.78) and ERK (62.24 � 1.16 and61.12 � 1.00) when compared with IL-1β alone (78.78 � 0.48and 71.80 � 2.21, respectively) (p < 0.05) (Figure 1), but not in adose-dependent manner. Similarly, an increased expression ofMMP-13 was observed with IL-1β stimulation (0.45 � 0.02)(p < 0.05) (Figure 1) that was inhibited by pretreatment with CT(50 and 500 ng/mL) (0.35 � 0.03 and 0.35 � 0.01, respectively),also not in a dose-independent manner (Figure 1). By comparison,pretreatment with CT (50 ng/mL and 500 ng/mL) did not sig-nificantly inhibit the phosphorylation levels of JNK (29.57 � 0.46and 29.48� 0.18) when compared with IL-1β alone (29.89� 0.32)(p > 0.05) (Figure 1), with CT pretreatment having.

Effects of CT on Cartilage Gross Morphology andHistopathologyA rat model of OA was induced by surgical transection of theanterior cruciate ligament, with a sham surgery as a control.Following this, rats were treated with CT or normal saline for a

period of 12 weeks to assess the protective effects of CT. Grossobservations showed that the sham-operated joint displayed asmooth cartilage surface. In contrast, the ACLT joint treatedwith saline displayed an ulcerated surface with the presence oflarge periarticular osteophytes. CT-treated ACLT joints, on theother hand, had a roughened surface, with slight ulcerations andsmall periarticular osteophytes (Figure 2A).

Histopathological analysis found that the sham-operativegroup showed a normal appearance in the cartilage joint. TheACLT þ NS group displayed significant degenerative character-istics, including an ulcerated surface, a loss of matrix staining, areduction in cartilage thickness and cell number, and cell cluster-ing. In the ACLT þ CT group, the joint showed a moderatedegeneration, including cartilage fissuring and fibrillation(Figure 2B).

TheMankin score reflects pathological changes to cartilage. Forthe ACLTþNS and ACLTþ CT groups, the Mankin scores werehigher than that in sham-operative group (p < 0.05) (Figure 2C).CT treatment reduced the Mankin score by 47% when comparedwith normal saline treatment in the respective ACLT groups.

Effects of CT on Cartilage Extracellular Matrix DegradationImmunohistochemistry analysis found a significant decrease intype II collagen expression and an increase in the expressions ofADAMTS-4 and MMP-3 in the ACLT þ NS and ACLT þ CTgroups when compared with that in the sham-operative group(p < 0.05) (Figure 3A and B). Interestingly, rats treated with CTshowed a 51% increase in type II collagen expression, a 42%decrease in ADAMTS-4, and a 39% decrease in MMP-3 whencompared with those measurements in the ACLT þ NS group(Figure 3A and B).

Effects of CT on Subchondral BMD DecreaseDXA scanning was performed in the subchondral ROI to deter-mine the BMD in the sham group versus the operated groupstreated with normal saline or CT. The results showed that theBMD in the ACLTþNS group was reduced by 11% in the medialcondyle and 5% in the lateral condyle when compared with thoseregions in the sham-operated group (Table 1). CT treatmentcaused an increase in these values by 12% in the medial condyleand 5% in the lateral condyle (Table 1).

Effects of CT on Subchondral Bone RemodelingGiemsa staining was used to determine the structural and dynamicparameters of the subchondral bone on undecalcified sectionsusing light and confocal microscopy. The results showed that thesubchondral trabecular bone was reduced in the ACLT groupwhen compared with that in the sham group (Figure 4). CTtreatment for 12 weeks alleviated the destruction to the subchon-dral bone following ACLT surgery (Figure 4).

Subchondral bone histomorphometric analysis revealed reduc-tions in BV/TV by 49%, Tb.Th by 39%, Tb.N by 25%, MS/BS by63%, MAR by 37%, and BFR/BS by 76% for rats in the ACLTgroup when compared with those parameters in the sham-operation group (Table 2). Tb.Sp in the ACLT group wasincreased by 103% when compared with that in the sham-operation group (Table 2). When compared with normal salinetreatment, CT significantly improved both the structural anddynamic parameters of the subchondral bone, increasing BV/TVby 44%, Tb.Th by 23%, Tb.N by 17%, MS/BS by 40%, MAR by34%, and BFR/BS by 83% and decreasing Tb.Sp by 28%, whencompared with the ACLT þ NS group (Table 2).

Potential Protective Effects of Calcitonin

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Discussion

The in vitro and in vivo findings in our study confirm that CTcounteracts the histomorphological cartilaginous deteriorationand subchondral bone loss associated with OA in a rat OAmodel. Importantly, we further found that CT inhibits IL-1β-mediated chondrocytes from synthesizing MMP-13 and regulatesthe macromolecular expression levels of key extracellular matrixcomponents (type II collagen, MMP-3, and ADAMTS-4). CT alsoaccommodates osteoclast-driven subchondral trabecular align-ments to correct for mechanical changes in the knee joints inducedby ACLT surgery.

Chondrocytes are solo functioning cells in articular cartilage andcan respond to inflammatory factors via various signaling pathways[17]. Evidence to date indicates that IL-1β is a potent stimulator thatincreases MMP-13 synthesis and secretion by chondrocytes, whichin turn can aggravate articular cartilage extracellular matrix degra-dation [18,19]. Furthermore, previous studies have shown that IL-1β-induced production of MMP-13 is mediated by ERK, p38kinase, and JNK components of the MAPK signal transductioncascade [20–22]. The MAPK family coordinates the expression ofmany genes that encode cytokines, chemokines, and other media-tors involved in the synthesis and further amplification of the

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Figure 1. Western blot analysis for the MAPK pathway and MMP-13 expression in IL-1β-induced chondrocytes. The expression of components of the MAPKpathway and MMP-13 were increased significantly in IL-1β-induced chondrocytes when compared with control. The protein expression levels of phospho-p38, phospho-ERK1/2, andMMP-13 after CT pretreatment were lower than that in IL-1β-induced cells, but there were no significant dose-dependent effects ofCT on the inhibition of phospho-p38, phospho-ERK1/2, and MMP-13. Western blot analysis showed that CT pretreatment did not affect phospho-JNKexpression in IL-1β-induced chondrocytes. In addition, CT pretreatment had no effect on MAPK pathway components in normal chondrocytes. Quantitativewestern blot measurements for phospho-p38, phospho-ERK1/2, andMMP- 13 showed that the integrated optical density (IOD) level in the high- and low-doseCT pretreatment groups were lower than that in the IL-1β-induced group.※, p < 0.05 versus control group.◆, p < 0.05 versus IL-1β-induced group.

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inflammatory reaction [23,24], each generating a distinct responseto the type of extracellular stimulus. However, our western blotresults demonstrated that pretreatment of chondrocytes with CTinhibited not only IL-1β-induced MMP-13 expression but also theprotein expressions of phospho-p38 and phospho-ERK. Therefore,our findings demonstrate, for the first time, that the chondropro-tective effect of CT in inhibiting IL-1β-induced MMP-13 proteinexpression was regulated by phospho-p38 and phospho-ERK path-ways in rat chondrocytes. In addition, we observed that there was noeffect of CT pretreatment on phospho-JNK expression in IL-1β-induced chondrocytes. It might be speculated that pretreatment ofCT could inhibit IL-1β-induced MMP-13 expression via the

inhibition of different upstream signaling molecules in the discreteMAPK pathways.

The normal extracellular matrix consists of water and a specificarrangement of macromolecules that provide the smooth cartilagesurface [25]. The breakdown of type II collagen is a key event inthe progression of OA that disrupts the collagen network andreduces the overall cartilage tensile strength [26]. Bagger et al.found that CT treatment reduced the level of CTX-II, a specialbiomarker of cartilage, both in experimental studies and in aclinical trial [27]. However, our immunohistochemistry resultsshowed that CT pretreatment in a rat ACLT-induced modelleads to a higher expression of type II collagen when compared

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ACLT + CTFigure 2. Gross appearance, histological find-ings, and Mankin score of articular cartilage ina rat osteoarthritis model at 12 weeks postsur-gery. (A) Sham-operated joints showed a smoothcartilage surface without osteophyte formationin the tibial plateaus and femoral condyles. In theACLT þ NS group, a marked increase in osteo-phyte formation (arrowhead) was observed onthe tibial plateau, and a large ulceration occurred(arrow) on the femoral condyle. In theACLT þ CT group, the tibial plateau showed asignificantly roughened cartilage surface, withosteophyte formation (arrowhead) at the mar-gins of the medial tibial plateau. A small ulcera-tion (arrow) was found on the femoral condyles.(B) Sham-operated joint showed a smootharticular cartilage surface stained with Safranin-O and HE staining. The ACLT joints displayed aloss of Safranin-O and HE staining, cell cloning,and surface fibrillation. In the ACLT þ CTgroup, the articular cartilage showed a slightlyroughened surface, with a mild loss of Safranin-O and HE matrix staining. (C) The cartilagelesions were graded byMankin scores, expressedas the mean � standard deviation.※, p < 0.05 versus Sham group.◆, p < 0.05 versus ACLT þ NS group.

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with normal saline treatment. Thus, CT directly contributes toextracellular matrix integrity. ADAMTS-4 and MMP-3 arethought to be important catabolic enzymes responsible for extra-cellular matrix turnover. ADAMTS-4 has been shown to be selec-tively overexpressed in human osteoarthritic cartilage, with itsexpression directly related to the degree of cartilage lesion, asassessed by the Mankin score [28]. Recent data have shown thatADAMTS-4 not only cleaves aggrecan but is also speculated tohave a wider proteolytic spectrum. MMP-3, on the other hand, isan interesting catabolic enzyme, which is produced by chondro-cytes and synovial cells [29,30]. A previous study showed thatMMP-3 plays an important role in rheumatoid arthritis [31],and a recent report by Lohmander et al. demonstrated thatMMP-3 acts as a potent predictor of OA when evaluating jointspace narrowing in the knees of patients [32]. Using ELISA assays,a clinical trial has found that an oral formulation of salmon CT in1 mL doses can reduce the serum and urinary levels of stromelysin1 (MMP-3) and collagenase 3 (MMP-13) [33]. The inhibition of

ADAMTS-4 or MMP-3 is considered to have a protective effect inthe maintenance of articular cartilage [34,35]. In line with theseassumptions, we have shown that CT treatment decreases theexpressions of ADAMTS-4 and MMP-3 in rats after ACLT sur-gery. The present findings clearly suggest that CT treatment, atleast in part, can protect the integrity of the extracellular matrix inthe early stages of OA.

Currently, subchondral bone is considered to be an interestingtarget in OA treatment [36,37]. In the present study, we used DXAto scan the subchondral regions of the proximal tibia and found adecrease in the BMD in rats following ACLT surgery. The sub-chondral bone is sensitive to micro-environmental changes in thejoint [38]. The balance in bone metabolism is disturbed by abnor-mal loadings that are induced by the ACLT surgery, which resultsin a cascading effect that increases bone resorption in the earlystages of OA development, preceding the onset of subchondralsclerosis [39–41]. Moreover, research has emphasized that thesubchondral bone quality depends not only on BMD but also onthe inner microstructural properties of the bone [42]. Throughmicroscopic observations, studies demonstrate that, in OA, sub-chondral trabecular micro-damage can trigger endochondral ossi-fication and aggravate cartilage degradation [43]. Thisaccumulated trabecular micro-damage is harmful to the boneremodeling system and destroys the subchondral trabecularmicrostructure. A recent study has indicated that the bone ana-bolic agent, parathyroid hormone, can preserve the trabecularmicrostructure and reduce cartilage damage in a rabbit model ofOA that is preceded by osteoporosis [44]. In a canine experimentalmodel of OA, the inhibitory effect of CT on the subchondral boneloss was considered to offer a protective effect to the cartilage [9].It is known that CT binds to CT receptors on osteoclasts andinhibits osteoclast-mediated bone metabolism. Our results con-firm that CT treatment not only inhibits the decrease in

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Figure 3. Immunohistochemical analysis of type IIcollagen, ADAMTS-4, and MMP-3 in articularcartilage. (A) Sham-operated cartilage showednor-mal expression of type II collagen in the cytoplasmof chondrocytes in the upper zone of cartilage,whereas the ADAMTS-4 and MMP-3 expressionswere hardly found in sham-operated cartilage. Inthe ACLT þ NS group, the impaired cartilageshowed a significant decrease in the expression oftype II collagen and amarked increase in the levelsof ADAMTS-4 and MMP-3. In the ACLT þ CTgroup, CT upregulated the expression of type IIcollagen and markedly inhibited the expressionlevels of ADAMTS-4 and MMP-3. (B) Thesechanges in expression were measured by IOD,which are expressed as themean� standard devia-tion (200� magnification).※, p < 0.05 versus Sham group.◆, p < 0.05 versus ACLT þ NS group.

Table 1. BMD of subchondral bone (g/cm2)

R

Group R1 R2

Sham 0.2327 � 0.0009 0.2345 � 0.0027ACLT þ NS 0.2081 � 0.0013* 0.2227 � 0.0027*ACLT þ CT 0.2228 � 0.0028*† 0.2282 � 0.0022*†

R, region; R1, medial femoral condyle; R2, lateral femoral condyle; ACLT, anteriorcruciate ligament transection surgery; NS, normal saline; CT, calcitonin. Results arethe mean � standard deviation.*p < 0.05 versus Sham group.†p < 0.05 versus ACLT þ NS.

Sham

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ACLT + NS ACLT + CT Figure 4. Subchondral bone changes in the rightproximal tibia of rats at 12 weeks after ACLTsurgery. Giemsa staining of the subchondralbone showed a destruction of the subchondraltrabecular structure in the ACLT þ NS groupwhen compared with that in the sham group. CTtreatment improved the structure of the sub-chondral bone in rats exposed to ACLT (100�magnification).

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subchondral BMD but also improves the subchondral trabecularmicrostructure in a rat model of OA.

Conclusions

In conclusion, our study demonstrates that pretreatment with CTexerts an inhibitory action on MMP-13 expression in articularchondrocytes under the influence of IL-1β. Furthermore, in OAchondrocytes, this inhibitory effect might be partly mediated bythe inhibition of components of the MAPK pathway, such as p38and ERK. In terms of extracellular matrix degradation, CT treat-ment regulates the expressions of type II collagen, MMP-3, andADAMTS-4, contributing to the histological integrity of cartilage.CT treatment also preserves the subchondral BMD and trabecularmicrostructure. Taken together, our findings confirm the protec-tive effect of CT on chondrocytes, the cartilaginous extracellularmatrix, and the subchondral trabecular bone in rats with experi-mentally induced OA.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of this article.

Grant supports were provided by National Natural ScienceFoundation of China (No. 31171136).

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Table 2. Bone histomorphometrical analysis of subchondral trabecular bone

Group Sham ACLT þ NS ACLT þ CT

BV/TV (%) 52.21 � 7.34 26.46 � 4.68* 38.03 � 2.72*†Tb.Th (μm) 67.93 � 8.26 46.21 � 9.25* 56.98 � 7.49*†Tb.N (#/mm) 7.81 � 1.00 5.84 � 0.77* 6.86 � 0.97*†Tb.Sp (μm) 63.67 � 17.82 129.51 � 21.80* 93.39 � 15.26*†MS/BS (%) 0.41 � 0.03 0.15 � 0.04* 0.21 � 0.02*†MAR (μm/d) 3.01 � 0.13 1.91 � 0.14* 2.56 � 0.10*†BFR/BS (μm/d � 100) 1.23 � 0.11 0.29 � 0.07* 0.53 � 0.04*†

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Potential Protective Effects of Calcitonin

© 2013 Informa Healthcare USA, Inc.

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