Research ArticleOsteoarthritis

Teriparatide as a Chondroregenerative Therapy for Injury-Induced Osteoarthritis

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Science Translational Medicine  21 Sep 2011:
Vol. 3, Issue 101, pp. 101ra93
DOI: 10.1126/scitranslmed.3002214

Abstract

There is no disease-modifying therapy for osteoarthritis, a degenerative joint disease that is projected to afflict more than 67 million individuals in the United States alone by 2030. Because disease pathogenesis is associated with inappropriate articular chondrocyte maturation resembling that seen during normal endochondral ossification, pathways that govern the maturation of articular chondrocytes are candidate therapeutic targets. It is well established that parathyroid hormone (PTH) acting via the type 1 PTH receptor induces matrix synthesis and suppresses maturation of chondrocytes. We report that the PTH receptor is up-regulated in articular chondrocytes after meniscal injury and in osteoarthritis in humans and in a mouse model of injury-induced knee osteoarthritis. To test whether recombinant human PTH(1–34) (teriparatide) would inhibit aberrant chondrocyte maturation and associated articular cartilage degeneration, we administered systemic teriparatide (Forteo), a Food and Drug Administration–approved treatment for osteoporosis, either immediately after or 8 weeks after meniscal/ligamentous injury in mice. Knee joints were harvested at 4, 8, or 12 weeks after injury to examine the effects of teriparatide on cartilage degeneration and articular chondrocyte maturation. Microcomputed tomography revealed increased bone volume within joints from teriparatide-treated mice compared to saline-treated control animals. Immediate systemic administration of teriparatide increased proteoglycan content and inhibited articular cartilage degeneration, whereas delayed treatment beginning 8 weeks after injury induced a regenerative effect. The chondroprotective and chondroregenerative effects of teriparatide correlated with decreased expression of type X collagen, RUNX2 (runt-related transcription factor 2), matrix metalloproteinase 13, and the carboxyl-terminal aggrecan cleavage product NITEGE. These preclinical findings provide proof of concept that Forteo may be useful for decelerating cartilage degeneration and inducing matrix regeneration in patients with osteoarthritis.

Introduction

Since 2005, arthritis has been ranked as the number one cause of disability in the United States (1). In 2008, 27 million Americans were afflicted by osteoarthritis (OA) (2), with 25% of the adult U.S. population (>67 million people) projected to have the disease by 2030 (3). Although there are numerous established causes of OA, the disease process is always characterized by progressive articular cartilage degeneration, subchondral sclerosis, and osteophyte formation, which culminates in complete loss of cartilage and bone-on-bone articulation (eburnation) during end-stage disease (46). Trauma that causes damage to the meniscus or ligaments within the knee is widely accepted as a common cause of OA in this joint (7), with an estimated sixfold increase in the risk of developing radiographic evidence of disease within 21 years after injury (8). Current treatments for OA are palliative, aimed at reducing pain, which is the main symptom of the disease (9). In advanced disease, the joint may be surgically replaced; however, this is a major surgical procedure with considerable morbidity as well as long-term consequences owing to joint failure and infection (9).

The articular chondrocyte is the cell responsible for maintenance of articular cartilage in adults (10). As opposed to chondrocytes associated with bone development and growth, articular chondrocytes do not normally undergo hypertrophic maturation leading to matrix mineralization. Instead, they persist in their expression of collagen types II, VI, IX, and XI; cartilage oligomeric matrix protein; and aggrecan, all of which are secreted proteins essential for the integrity of articular cartilage (11). In the context of OA, articular chondrocytes begin expressing genes associated with maturation during endochondral ossification, including type X collagen, alkaline phosphatase, and RUNX2 (runt-related transcription factor 2). Catabolic enzymes, including matrix metalloproteinases 9 and 13 (MMP9 and MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS4 and ADAMTS5), are also up-regulated (1115). Biochemical, genetic, and mechanical factors are likely contributors to this change in cell phenotype, which culminates with degeneration of cartilage in OA. Thus, strategies to inhibit aberrant hypertrophic maturation and to induce matrix production in articular chondrocytes could represent potential new therapeutic modalities.

There is an abundance of literature documenting the stimulatory effects of parathyroid hormone (PTH) on bone formation (1618). This work led to the development of Forteo (Eli Lilly), the Food and Drug Administration (FDA)–approved formulation of recombinant human PTH(1–34) (teriparatide), which is a bone anabolic therapy for osteoporosis (19). Although the mechanism underlying the anabolic action of teriparatide treatment on bone is not fully understood, direct effects on osteoblast proliferation and survival are likely important (16, 20, 21). In addition to bone as a target, signaling downstream of the type I parathyroid hormone receptor (PTHR1) regulates chondrocyte differentiation (22, 23). Parathyroid hormone–related peptide (PTHrP), another PTHR1 ligand, is a potent stimulator of proliferation and matrix synthesis and suppressor of maturation (24, 25). Mice that are homozygous for the ablation of Pthrp (26) or Pthr1 (27) display accelerated chondrocyte maturation and premature bone formation. Conversely, animals overexpressing PTHrP in the growth plate display delayed terminal differentiation and a disruption of normal mineral deposition (28). Chondrocyte-targeted overexpression of the constitutively active PTHR1 mutant that causes Jansen’s metaphyseal chondrodysplasia also induces disorganization of the growth plate with delayed terminal differentiation (29). These in vivo data [reviewed in (30, 31)] collectively implicate PTHR1 signaling as a key regulator of chondrocyte differentiation and endochondral ossification.

PTHrP is normally expressed by articular chondrocytes in low amounts and is increased after application of mechanical load (32, 33) or in OA (34). It has been established recently that PTHrP is required for articular cartilage maintenance given that Gdf5-Cre–targeted knockdown of PTHrP in mid-region articular chondrocytes leads to accelerated development of posttraumatic OA in the mouse (35). Along with data presented in this report, there is also evidence that PTHR1 is up-regulated in osteoarthritic cartilage (36). Given the proliferative and matrix anabolic effects of PTHR1 signaling in chondrocytes and the requirement for PTHrP in the maintenance of articular cartilage, the up-regulation of PTHR1 in articular chondrocytes during OA might represent an endogenous attempt at repair. Based on this, we designed experiments to test the hypothesis that teriparatide may have therapeutic potential for treating OA. To test this hypothesis, we examined the ability of systemic, intermittent teriparatide treatment to decelerate cartilage degeneration, and stimulate matrix synthesis in an established mouse meniscal/ligamentous injury (MLI) model of knee OA (37, 38).

Results

PTHR1 is up-regulated in OA cartilage and after meniscal injury

To examine PTHR1 expression in articular chondrocytes after meniscal injury or in progressive OA in humans, and after MLI in mice, we examined protein and mRNA levels using immunohistochemistry and quantitative polymerase chain reaction (qPCR), respectively. Normal human articular cartilage was obtained from amputation patients, and injured cartilage was obtained from patients undergoing arthroscopic treatment of meniscal injuries that involved articular cartilage debridement. Representative immunohistochemistry revealed that although PTHR1 was not detectable in articular chondrocytes in normal cartilage (Fig. 1A), cellular expression of PTHR1 was observed in cartilage from human patients with meniscal injury (Fig. 1B) or with progressive OA (Fig. 1C). The expression pattern was similar in the mouse, with increased PTHR1 expression in articular chondrocytes from MLI joints (Fig. 1E, red arrows) compared to sham-operated joints (Fig. 1D). As expected, osteoblasts and osteocytes in the subchondral bone were PTHR1-positive in both control and MLI joints (Fig. 1, D and E, black arrows). Consistent with this, qPCR analysis of mRNA harvested from articular cartilage revealed that Pthr1 mRNA expression was fivefold greater in MLI versus sham joints (Fig. 1F, P < 0.05). Mmp13 and Adamts5, transcripts for enzymes responsible for the degradation of type II collagen and proteoglycans, respectively, were also up-regulated in injured joints compared to controls (Fig. 1F), confirming a progressive degenerative process after MLI. These results establish that PTHR1 is increased in both human and mouse articular cartilage after meniscal injury and in progressive human OA, priming articular chondrocytes to be responsive to endogenous PTHrP as a repair response. In this context, PTHR1 might represent a therapeutic target for systemically delivered exogenous teriparatide.

Fig. 1

PTHR1 is increased in human and murine articular cartilage after injury and during OA. (A to C) Staining for PTHR1 in articular chondrocytes in normal human cartilage (A), cartilage from patients who underwent arthroscopic surgery for a meniscal injury (MI) (B), and within OA cartilage from patients who underwent total knee arthroplasty (C). Blue scale bar in (A), 100 μm [applicable to (A) to (C)]. (D and E) PTHR1 expression in articular chondrocytes within sham-operated, uninjured mouse knee cartilage (D) and arthritic cartilage from mice with MLI injury (E, red arrows). Osteoblasts and osteocytes in the subchondral plate positive for PTHR1 are indicated by black arrows. Red scale bar in (D), 25 μm [applicable to (D) and (E)]. (F) qPCR analysis of mRNA of chondrocytic markers isolated from the tibial plateau articular cartilage of mice that underwent sham and MLI surgery. *P < 0.05 compared to respective sham controls using an unpaired, two-tailed Student’s t test. Bars represent means ± SEM (n = 3).

Systemic administration of Forteo has anabolic effects on bone

The bone anabolic effect of teriparatide was confirmed by microcomputed tomography (μCT) analysis and detection of type I collagen (Col1a1) expression via in situ hybridization. Sham-operated and MLI mice were administered either saline or Forteo (40 μg/kg per day), commencing after MLI surgery (immediate) or 8 weeks later (delayed). Forteo treatment lasted either 12 weeks for the “immediate” group or 4 weeks for the “delayed” group. In sham-operated mice, bone volume was greater in Forteo-treated mice compared to saline-treated controls (Fig. 2A, immediate and delayed), confirming that both administration regimens induce a bone anabolic effect similar to that published (16). In injured mice, there was an increase in bone volume in both the saline-treated groups (Fig. 2B, immediate and delayed) compared to saline-treated, sham-operated controls (Fig. 2A, immediate and delayed), which is consistent with the development of a sclerosis in the subchondral plate that is associated with the OA degenerative process seen in humans (39). In MLI mice, both immediate and delayed treatment of MLI mice with teriparatide increased bone volume significantly compared to the saline-treated MLI groups (Fig. 2B, P < 0.05). This increased bone volume with delayed teriparatide treatment in MLI mice was associated with increased expression of Col1a1 by cells in the subchondral bone (Fig. 2, C and D), further confirming the bone anabolic effects of systemic teriparatide treatment in the joint region.

Fig. 2

Systemic Forteo treatment has a bone anabolic effect and induces articular chondrocyte up-regulation of JAG1 in MLI joints. (A and B) μCT scans were performed on sham-operated and MLI mouse joints 12 weeks after surgery, and bone volume in the region between the physes was quantified. The median bone volume for each experimental group is indicated by the horizontal line within each box plot, with the top and bottom of each box indicating the 75th and 25th percentile, respectively (n = 10). Whiskers denote maximum and minimum data points in the set. *P < 0.05 compared to respective sham controls using ANOVA. (C to F) Eight weeks after sham or MLI surgery, either saline or Forteo was administered to the mice daily for 4 weeks (delayed administration). Representative sections are shown hybridized with a probe specific for Col1a1 in the subchondral bone of MLI joints from Forteo-treated mice (D) and from saline-treated mice (C). Representative unstained sections from these same joints were also probed with a JAG1 antibody to reveal immunoreactivity in articular chondrocytes (red arrows) (E and F). Red scale bar in (C), 25 μm [applicable to (C) to (F)].

Forteo increases JAGGED1 expression in articular chondrocytes after MLI

JAGGED1 (JAG1) immunohistochemistry was performed to confirm that systemic administration of teriparatide leads to cartilage-specific responses in injured joints. JAG1, a membrane-bound ligand in the Notch signaling pathway, was chosen as a readout of receptor activation because of its direct transcriptional activation in response to PTHR1 signaling in several cell types, including osteoblasts and periodontal ligament cells (40). Beginning 8 weeks after MLI surgery (delayed administration), mice were treated with either saline or Forteo for 4 weeks, and joints were harvested. Joints from Forteo-treated MLI mice showed a greater amount of articular chondrocyte JAG1 immunoreactivity (Fig. 2F, red arrows) compared to saline-treated controls (Fig. 2E), establishing that the systemic, intermittent treatment regimen delivers Forteo to the joint where it can exert a direct effect on articular chondrocytes.

Teriparatide enhances proteoglycan content during early degeneration in MLI joints

To determine whether teriparatide induction of PTHR1 signaling in articular chondrocytes influences the progression of injury-induced cartilage degeneration, we treated mice administered sham surgery or MLI immediately with saline, Forteo, or PTH, and harvested joints 4 weeks later. Histomorphometry was performed to quantify cartilage area, and Alcian Blue staining was used to assess proteoglycan content in the articular cartilage. At this early time point, histological analysis revealed only mild arthritic changes in injured joints, notably fibrillation (fig. S1, D to F, black arrows). No difference in articular cartilage area in sham versus injured joints was discernable by quantitative histomorphometry (fig. S1G). The up-regulation of PTHR1 mRNA and protein 8 weeks after MLI (Fig. 1) suggested that articular chondrocytes acquired sensitivity to teriparatide before substantial articular cartilage degeneration. Although neither Forteo nor PTH treatment affected articular cartilage area in injured joints at this early time point, there was increased articular cartilage Alcian Blue staining intensity in treated mice (fig. S1G), suggesting that teriparatide induces proteoglycan synthesis in articular chondrocytes in cartilage that is beginning to undergo injury-induced osteoarthritic degeneration. There was no apparent difference in articular cartilage Alcian Blue staining intensity in sham-operated joints in mice treated with saline versus Forteo or PTH (fig. S1, A to C), which confirms that teriparatide only stimulates proteoglycan matrix production in articular chondrocytes with increased PTHR1 after MLI.

Immediate treatment with Forteo inhibits articular cartilage loss after MLI

By 12 weeks after surgery in saline-treated mice, there was extensive articular cartilage degeneration in MLI joints (Fig. 3C) compared to sham-operated joints (Fig. 3A). Comparatively, although immediate treatment of sham-operated mice with Forteo did not alter cartilage morphology (Fig. 3B), it decelerated cartilage degeneration in MLI joints (Fig. 3D). Mouse Osteoarthritis Research Society International (OARSI) scoring of joint cartilage revealed a significantly better score in injured joints of mice administered Forteo than in saline-treated controls (Fig. 3E). Moreover, histomorphometric analysis of MLI joints revealed that Forteo treatment inhibited the OA-like decrease in articular cartilage area between the 8- and 12-week time points seen in saline-treated mice, evidenced by 27% more articular cartilage area after Forteo versus saline treatment 12 weeks after injury (Fig. 3F, P < 0.05). These results establish that immediate systemic treatment with teriparatide after MLI in mice is chondroprotective, supporting it as a candidate therapeutic agent that can suppress cartilage loss in OA, which is the central hallmark of the degenerative process in human disease.

Fig. 3

Immediate treatment with Forteo protects against articular cartilage degeneration in MLI joints. Mice underwent MLI (right knee) and sham (left knee) surgery and then immediately began daily systemic saline or Forteo treatment for 12 weeks. (A to D) Representative Alcian Blue/Orange G staining for cartilage degeneration after MLI or sham surgery in immediate saline-treated (A and C) and immediate Forteo-treated (B and D) animals. In (C), red arrows identify areas of fibrillation and clefting and the black arrow identifies eburnation (erosion to subchondral bone). Black scale bar in (A), 50 μm [applicable to (A) to (D)]. (E and F) Chondroprotective effects of immediate Forteo treatment were confirmed by mouse OARSI scoring (E) and by cartilage area determination (F). *P < 0.05 compared to respective saline-treated MLI or sham controls using an unpaired, two-tailed Student’s t test. Bars represent means ± SEM. The white hatched bar and red line indicate the average normalized cartilage area in joints 8 weeks after MLI in saline-treated animals.

Delayed treatment with Forteo induces articular cartilage regeneration after injury

To examine the effect of Forteo on cartilage that is progressively degenerating, we initiated a 4-week treatment period with saline or Forteo 8 weeks after sham surgery or MLI. This delayed regimen was used to examine the impact of treatment in the context of the clinically relevant situation where the therapy would be initiated after a diagnosis of OA during the progressive degeneration stage. Representative histological sections from joints 12 weeks after injury surgery (4 weeks after initiation of treatment) showed that saline-treated MLI mice displayed substantial cartilage loss that involved exposure of the subchondral bone in some areas (Fig. 4C), which is in contrast to the intact articular cartilage layer seen in sham-operated joints (Fig. 4A). Forteo-treated MLI mice (Fig. 4D) displayed a larger amount of cartilage than saline-treated MLI mice (Fig. 4C), a finding that was consistent with the effect of immediate teriparatide treatment seen in Fig. 3. Mouse OARSI scoring of the joints supported these histological results, with significantly lower scoring of the Forteo-treated MLI joints compared to the saline-treated control group (Fig. 4E, P < 0.05).

Fig. 4

Delayed Forteo treatment induces chondroregeneration in MLI joints. Eight weeks after MLI or sham surgery, mice began receiving daily systemic saline or Forteo treatment for 4 weeks. Joints were collected at week 12 for histological analysis. (A to D) Representative Alcian Blue/Orange G staining for cartilage degeneration after MLI or sham surgery in delayed saline-treated (A and C) and delayed Forteo-treated (B and D) animals. In (C), red arrows identify areas of fibrillation and clefting and black arrows identify areas of cartilage erosion and eburnation. Black scale bar in (A), 50 μm [applicable to (A) to (D)]. (E to G) Mouse OARSI score (E), Alcian Blue staining normalized to the sham control group (F), and cartilage area histomorphometry normalized to the sham control group (G) collectively establish effects of Forteo on cartilage integrity. *P < 0.05 compared to respective sham controls using an unpaired, two-tailed Student’s t test. Bars represent means ± SEM (n = 5).

To investigate whether teriparatide induces cartilage matrix production, we quantified Alcian Blue stain in histological sections to evaluate proteoglycan content. Image analysis revealed a nearly threefold increase in Alcian Blue–stained matrix in delayed Forteo-treated MLI joints compared to the saline-treated MLI controls (Fig. 4F, P < 0.05). This was consistent with the effect of teriparatide at 4 weeks after MLI when administered immediately, where a more than threefold increase in proteoglycan staining was also observed (fig. S1). Histomorphometric analysis of cartilage area supported this by establishing that the delayed Forteo-treated groups had significantly more cartilage than both saline-treated mice at 12 weeks after MLI (32% increase) and saline-treated mice at 8 weeks after MLI (21% increase) (Fig. 4G, P < 0.05). Overall, the increased proteoglycan matrix staining and increased cartilage area compared to that seen at 8 weeks after MLI suggest that systemic Forteo treatment has a chondroregenerative effect in degenerating cartilage.

To establish that the observed effects of Forteo are not specific to the clinical Forteo formulation that was administered to the injured mice, we repeated this experiment using recombinant human PTH(1–34). As done previously with Forteo, a 4-week treatment period with saline or PTH was initiated 8 weeks after administration of sham surgery or MLI. Representative histological sections from joints 12 weeks after surgery (4 weeks after initiation of treatment) showed the expected degeneration after MLI (fig. S2, A and C), with PTH-treated mice (fig. S2D) displaying a larger amount of cartilage than saline-treated mice (fig. S2C). Mouse OARSI scoring of the joints supported this observation, with significantly lower scores for PTH-treated MLI joints compared to the saline-treated control group (fig. S2E). Imaging to quantify proteoglycan content revealed nearly threefold more Alcian Blue–stained matrix in PTH-treated MLI joints compared to saline-treated MLI control joints (fig. S2F). Finally, histomorphometric analysis of cartilage area established that the PTH-treated groups had significantly more cartilage than both saline-treated mice at 12 weeks after MLI (24% increase; fig. S2G) and saline-treated mice at 8 weeks after MLI (13%; fig. S2G, blue bar and red line). These results establish that generic recombinant human PTH(1–34) induces the same chondroregenerative effect as Forteo.

Finally, the observed chondroregenerative effect of delayed Forteo administration correlated with increased expression of Proteoglycan4 (Prg4) mRNA by articular chondrocytes in MLI joints as determined by in situ hybridization. In a representative section from a sham-operated joint (Fig. 5A), Prg4, whose gene product is necessary for proper joint lubrication and function (41, 42), was expressed in the articular cartilage (red arrows) and posterior medial meniscus (blue arrows). Prg4 expression was lost in articular chondrocytes in MLI joints from saline-treated mice, although expression in the nonsurgically manipulated posterior medial meniscus was maintained (Fig. 5B, blue arrows). Delayed Forteo treatment restored the expression of Prg4 in articular chondrocytes in degenerating cartilage (Fig. 5C, red arrows), providing further evidence of a chondroregeneration. Notably, Forteo did not increase the expression of Prg4 in the meniscus (Fig. 5, B and C, blue arrows), indicating a selective effect of the therapy in degenerating cartilage that is populated by PTHR1-expressing articular chondrocytes (Fig. 1). Overall, teriparatide induces a chondroregenerative effect as evidenced by increased synthesis of critical matrix components, including Prg4, in degenerating cartilage. These results further establish a clinically relevant therapeutic potential for this agent in treating progressive OA.

Fig. 5

Delayed Forteo treatment induces articular chondrocyte Prg4 expression in MLI joints. (A to C) Prg4 expression (red arrows) in articular chondrocytes from sham-operated (A) and MLI joints (B and C), assessed via in situ hybridization in sections cut from the same experimental groups depicted in Fig. 4 (delayed Forteo treatment). Blue arrows identify areas of Prg4 expression in menisci. Black scale bar in (A), 50 μm (applicable to all panels).

Forteo treatment inhibits articular chondrocyte maturation and cartilage matrix degradation

It is hypothesized that OA pathophysiology stems, in part, from inappropriate endochondral ossification-like articular chondrocyte maturation (15, 43). Therefore, effective therapeutic strategies should aim to inhibit this aberrant maturation. We examined the expression of type X collagen (COL10a1) and RUNX2 in normal and arthritic joints harvested from mice administered saline or Forteo after MLI or sham surgery. Suggesting that injury-induced degeneration is associated with maturation of articular chondrocytes, MLI joints displayed enhanced COL10a1 levels (Fig. 6B) and increased cellular expression of RUNX2 (Fig. 6E, red arrows) compared to sham-operated joints (Fig. 6, A and D, respectively). Regarding the COL10a1 expression pattern, it was mainly restricted to the most hypertrophic cells in the deep layers of the cartilage in the sham-operated, saline-treated group (Fig. 6A, red arrows), whereas the more robust cellular and matrix staining in the arthritic joints was located near the cartilage surface (Fig. 6B, red and blue arrows, respectively). Consistent with the known inhibitory effect of PTHR1 activation on chondrocyte maturation (2225), injured joints from mice treated with Forteo showed significantly reduced cellular and matrix COL10a1 levels in the noncalcified articular cartilage (Fig. 6C), as well as reduced cellular expression of RUNX2 (Fig. 6F), compared to saline treatment (Fig. 6, B and E, respectively).

Fig. 6

Forteo inhibits chondrocyte maturation and reduces MMP13 levels in MLI joints. (A to F) Immunohistochemistry of cellular/matrix COL10a1 (A to C) and cellular RUNX2 (D to F) protein levels in MLI joints and sham controls. Red arrows indicate cellular staining and blue arrows indicate matrix staining. (G to I) Articular chondrocyte expression of MMP13 in MLI joints (H and I) and sham controls (G). (J to L) Immunohistochemical detection of the aggrecan cleavage product containing the C-terminal neoepitope NITEGE in matrix (blue arrows) and cells (red arrows) in the meniscus (M) and tibial plateau articular cartilage (T) from MLI joints (K and L) and sham controls (J). Red scale bar in (A), 25 μm (applicable to all panels).

In addition to inhibiting the expression of genes associated with chondrocyte maturation, Forteo reduced the degradation of matrix in joints after MLI. Consistent with mRNA results presented in Fig. 1F, protein levels of the catabolic enzyme MMP13 were increased in injured joints from saline-treated mice (Fig. 6H, red arrows) relative to sham-operated joints (Fig. 6G). In MLI joints from mice treated with Forteo (Fig. 6I), MMP13 levels were reduced compared to the saline-treated group (Fig. 6H). Regarding proteoglycan degradation, immunostaining for the C-terminal aggrecan cleavage product NITEGE revealed the expected increase in aggrecan matrix degradation in MLI joints (Fig. 6K) compared to sham-operated control joints (Fig. 6J). Red arrows denote enhanced cellular NITEGE staining in the meniscus and tibial plateau articular cartilage, and blue arrows identify areas of cartilage matrix with enhanced NITEGE staining. Suggesting inhibition of aggrecan breakdown in Forteo-treated MLI mice, NITEGE staining was reduced (Fig. 6L) compared to saline-treated MLI mice (Fig. 6K). These results establish that systemically administered Forteo reduces MMP13 levels and inhibits aggrecan degradation, suggesting the potential efficacy of teriparatide-based therapy in the inhibition of matrix catabolism that is central to the tissue phenotype present in the context of injury-induced OA.

Teriparatide treatment does not enhance osteophyte formation

A key feature of human OA is the development of osteophytes in the joint margins (39). Because PTHR1 signaling induced by Forteo leads to bone anabolic effects (Fig. 2, A and B), we examined whether MLI-induced osteophyte formation was exacerbated in MLI mice treated with either Forteo or PTH. Three-dimensional reconstructions were generated from the same μCT data sets that were collected for the morphometric data (Fig. 2, A and B). As depicted in the representative reconstructions, sham-operated mice treated with saline or Forteo did not display any sites of ectopic bone formation reminiscent of osteophytes at 12 weeks after surgery (fig. S3, A and B). As expected, MLI joints from mice treated with saline for 12 weeks showed significant mineral deposition at the joint margins and had enhanced mineralization of the meniscus (fig. S3C). Forteo-treated mice did not show enhanced osteophyte formation in the injured joints (fig. S3D) compared to the saline-treated control mice (fig. S3C). This finding was confirmed by histologic assessment of osteophyte number and mean diameter at the anterior and posterior margins of the tibial plateau. Images from representative sham-operated or MLI joints from mice given delayed saline or Forteo indicate that no osteophytes were initiated in sham-operated joints regardless of treatment (fig. S3, E and F). Comparatively, representative histology of MLI joints from mice treated with either saline or Forteo did reveal osteophytes in the anterior aspect of the medial compartment (fig. S3, G and H, blue arrows). Furthermore, neither Forteo nor PTH treatment caused an increased osteophyte number or diameter (fig. S3, I and J), with Forteo actually reducing osteophyte mean diameter in MLI joints from mice treated using the delayed regimen (fig. S3J). Overall, these results suggest that teriparatide therapy does not induce or exacerbate osteophyte formation that is naturally initiated by the degenerative process in this model.

Discussion

The most routinely recommended treatments for OA, including orally administered or locally injected anti-inflammatory agents and analgesics, are palliative, with surgical joint replacement the only option in advanced disease (9). The only therapies purported to be disease-modifying aim to replenish cartilage proteoglycan components via dietary supplementation with chondroitin sulfate/glucosamine (44, 45) or via intra-articular injection of hyaluronic acid (for example, Synvisc) (46, 47). However, there is no consensus on the efficacy of oral ingestion of aggrecan sugar moieties (45), and joint injections of hyaluronic acid demonstrate efficacy at relieving knee joint pain, but only for brief periods of up to 6 months (47). Therefore, the development of an effective agent supporting protective and/or regenerative effects in articular cartilage would have an immediate and major impact on standard of care for this pervasive and debilitating disease. Our findings establish teriparatide, in particular the FDA-approved drug Forteo, as a disease-modifying candidate therapeutic that has both chondroprotective and chondroregenerative capabilities in the context of OA.

At a cellular level, it has been suggested that aberrant maturation of articular chondrocytes along a pathway that resembles endochondral ossification contributes to OA progression (15, 43). This inappropriate articular chondrocyte differentiation is associated with the up-regulation of RUNX2 (48), type X collagen (12, 13), and MMP13 (43, 49), as well as increased apoptosis (14) and other hallmarks of chondrocyte maturation (15), in both human OA and animal models of disease. Consistent with this, in our mouse model of MLI-induced OA, we observed the up-regulation of Adamts5, COL10a1, RUNX2, MMP13 message and protein, and increased degradation of aggrecan in degenerating articular cartilage.

Signaling pathways inducing chondrocyte maturation and OA-like cartilage degeneration include loss of transforming growth factor–β (TGF-β) signaling in mice (5052), gain of WNT/β-catenin signaling in mice (53, 54) and humans (5557), gain of Indian Hedgehog (IHH) signaling in mice and humans (58), and increased hypoxia-inducible factor 2α (HIF2α) expression leading to enhanced IHH/RUNX2 signaling in mice (59, 60). These promaturation shifts in articular chondrocyte signaling are generally consistent with the additional findings that RUNX2 is up-regulated in injury-induced murine knee OA (48), and overexpression of RUNX2 in articular chondrocytes that are experiencing mechanical stress contributes to the pathogenesis of OA (61). These and other similar findings implicate maturation-driving signals in the pathogenesis of OA.

Modulation of signaling pathways that activate chondrocyte maturation might be protective against cartilage degeneration. For example, Runx2-haploinsufficient mice administered a meniscal injury display less severe knee OA (48). Similarly, pharmacologic and genetic inhibition of IHH signaling in mice protects against injury-induced degeneration of knee cartilage that is concomitant with down-regulation of Runx2 and ADAMTS5 (58). Furthermore, inhibition of cartilage-degrading enzymes associated with chondrocyte maturation, such as MMP13 and ADAMTS5, has been underscored as the best current strategic direction for developing an OA therapeutic (62). Thus, inhibition of inappropriate articular chondrocyte maturation and/or blockade of the associated matrix-degrading enzymes represent obvious targets for developing a treatment for OA.

It is well established that PTH and PTHrP are potent inhibitors of chondrocyte maturation (22, 23). Specifically, activation of PTHR1 potently induces chondrocyte proliferation and matrix production (type II collagen and proteoglycans) while suppressing maturation (2325). This concept is supported by in vitro and in vivo data, where gain or loss of PTHR1 signaling respectively inhibits or accelerates chondrocyte hypertrophy (30, 31). Owing to selective up-regulation of PTHR1, which we identified in human cartilage after meniscal injury or in progressive OA and in mouse cartilage after MLI, PTHR1 signaling may be protective and possibly even regenerative in the context of cartilage degeneration. This idea is supported by two studies, which demonstrate that when administered intermittently via intra-articular injection, PTH can decelerate papain-induced cartilage degeneration in the rat (63) and can induce cartilage regeneration in a full-thickness osteochondral injury in the rabbit (64). Although these studies suggest that PTH has chondroregenerative potential, the models of cartilage degeneration and injury that were used do not examine the effect of PTH during the OA disease process. Additionally, the daily intra-articular injection treatment regimen used in these studies is not clinically practical nor translational, leaving open the need to examine the efficacy of the systemic mode of delivery that is currently FDA-approved for teriparatide. Therefore, the rationale for the present investigation of teriparatide as a potential systemic OA therapy was based on two key concepts: the broad literature, which establishes PTHR1 signaling in chondrocytes as an inducer of matrix production and inhibitor of maturation, and the selective up-regulation of PTHR1 after injury and in arthritic cartilage, which primes the cells to be targeted by teriparatide therapy.

The experimental design to test teriparatide as an OA therapy involved treatment of mice administered a meniscal/ligamentous knee injury followed by treatment with either Forteo or PTH at a dose of 40 μg/kg per day. This dose was selected based on literature establishing that the effective teriparatide dose range for treatment of bone loss or fracture repair in mice is between 30 and 400 μg/kg per day (16, 6568). In two of these studies, 40 μg/kg per day was demonstrated to effectively accelerate fracture healing in the mouse, with enhancement of chondrogenesis and expansion of the cartilaginous callus (65, 66). Note that 40 μg/kg per day is significantly higher than the optimal ranges reported in other species including rat (4 to 20 μg/kg per day) (69, 70), rabbit (10 μg/kg per day) (71), macaque (5 μg/kg per day) (72), and human (0.25 μg/kg per day) (73). We anticipate that there is a similar species-associated shift in the range of effective concentrations supporting chondroprotective and chondroregenerative effects.

Compared to saline treatment and when delivered immediately after injury, Forteo was chondroprotective, characterized by enhanced proteoglycan production by 4 weeks and decelerated cartilage degeneration at 12 weeks. Notably, the more clinically relevant delayed treatment regimen elicited a chondroregenerative and maturation-inhibiting effect, characterized by increased proteoglycan content in the articular cartilage, up-regulation of Prg4, an increased amount of articular cartilage, and decreased articular chondrocyte expression of COL10a1, RUNX2, and MMP13. Degeneration of aggrecan was also reduced. Chondroregenerative effects were also observed when injured mice were treated with PTH, excluding the possibility that the effects are unique to the Forteo formulation. Ameliorating the concern that teriparatide might exacerbate osteophyte formation in degenerating joints, μCT analyses and histological measurement of osteophyte number and diameter establish that osteophytes were not increased in injured joints from Forteo-treated mice.

Histological and histomorphometric data suggest that inhibition of matrix-degrading enzymes may be more effective than teriparatide therapy at stopping degeneration of cartilage matrix during OA (74, 75). Specifically, compared to joints from injured mice treated with Forteo or PTH in the present study, the articular cartilage is better preserved after injury induction of OA in Adamts5 (74) and Mmp13 (75) knockout mice. However, the genetic ablation of catabolic enzymes in these studies leads to complete arrest of matrix degeneration without affecting other changes associated with disease, including aberrant chondrocyte maturation, osteophyte formation, and subchondral sclerosis. This is in contrast to the chondroregeneration and inhibition of chondrocyte maturation seen in the present study after teriparatide administration, which suggests that this therapy may be effective at decelerating several key disease phenotypes in addition to cartilage degeneration. Nevertheless, the chondropreservation observed in the Adamts5 and Mmp13 knockout mice has led to the conclusion that inhibitory agents targeting these enzymes have therapeutic potential (62). Initial studies in the rat have substantiated the in vivo efficacy of an aggrecanase inhibitor in the blockade of aggrecan degradation (76) and MMP inhibitors in decelerating cartilage degeneration (77, 78). The only way to elicit similar cartilage preservation clinically would be to administer enzyme inhibitors before degeneration begins and to persist with the treatment over the span of a lifetime. To begin treatment when a patient presents to the clinic with pain because of progressed disease would potentially prevent further degeneration, but it would not have a reparative effect that would build back matrix. A potential strategy for more effective therapeutic results would be to combine periodic teriparatide treatment with the administration of an ADAMTS or MMP inhibitor; this could lead to even more robust chondroregeneration by teriparatide owing to the complementary blockade of matrix degradation.

Although teriparatide elicits a bone anabolic effect in the subchondral plate via activation of osteoblasts, it might simultaneously restrict the endochondral ossification-like process occurring in MLI-associated osteophytes. This would occur via inhibition of chondrocyte maturation in a manner analogous to the action of PTHrP in the developing growth plate. This possibility is supported by our findings that Forteo inhibits expression of genes associated with chondrocyte maturation in degenerating cartilage. Regarding the increased bone volume in the subchondral plate in both MLI and sham-operated joints after Forteo treatment, there is a concern that this effect might enhance subchondral sclerosis and exacerbate, rather than decelerate, the cartilage degeneration that occurs in clinical OA. This concern, coupled with practical considerations regarding the persistence of the chondroprotective or chondroregenerative effects of teriparatide after the treatment is stopped, represents key questions to be addressed.

There is evidence for teriparatide induction of osteosarcoma in rats administered long-term daily treatment out to 2 years, with a 20 to 40% incidence at 70 to 80% of life span (79). In our examination of articular cartilage in samples harvested from teriparatide-treated mice, we never observed a lesion consistent with a malignancy in the tibial or femoral metaphyses. Because these locations are among the most common sites for primary osteosarcoma, we do not believe that our immediate or delayed treatment regimens are carcinogenic in the mouse. This is consistent with what has been reported in macaques, where teriparatide did not induce any osteosarcomas after long-term treatment with 5 μg/kg per day (72). Furthermore, human data suggest that only 2 cases in >430,000 osteoporosis patients treated with Forteo developed osteosarcoma (73). Thus, we believe that the increase in osteosarcoma incidence in teriparatide-treated rats probably is not prognostic of an equivalent risk in humans (80) and is possibly species-specific.

In conclusion, we have identified teriparatide as a novel candidate therapy for injury-induced OA that is both chondroprotective and chondroregenerative. This affects the arthritis field by providing the basis for FDA-approved Forteo as a disease-modifying therapy for OA with clinical potential. Overall, given the scope of the clinical problem and the current availability of Forteo for clinical use, our experimental findings make a compelling case for further characterization of this drug in a clinical trial aimed at evaluating its efficacy as a treatment for human OA.

Materials and Methods

Procurement and fixation of human tissues

An Institutional Review Board–approved protocol was executed to collect discarded cartilage from patients undergoing orthopedic surgery. Normal cartilage was collected from amputation patients (talus or knee, n = 2), damaged cartilage was debrided from patients who underwent arthroscopic surgery to treat a meniscal injury (n = 18), and OA cartilage was harvested from patients who underwent total knee arthroplasty (n = 22). Tissues were fixed for 2 to 10 days in 10% neutral buffered formalin at 25°C. Samples were decalcified for 3 weeks in 10% (w/v) EDTA and then embedded in paraffin. Sections (3 μm) were cut and mounted on positively charged slides, baked at 60°C for 30 min, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol.

OA-inducing MLI surgery and teriparatide treatment

All experiments involving mice were performed with the approval of the University Committee on Animal Resources at the University of Rochester Medical Center. Ten-week-old male C57BL/6 mice were administered MLI to the right knee and sham surgery to the left knee by a surgical method that we established previously (38). After administration of anesthesia [intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg)], a 5-mm-long incision was made on the medial aspect of the joint. With the aid of surgical loops, the medial collateral ligament was transected, the joint space was opened slightly, and a 25-gauge needle was used to detach the medial meniscus from its anterior attachment to the tibia. Scissors were then used to remove a portion of the detached meniscus. Sham surgery involved a similar incision to open, but tissues were not manipulated. After this, the skin was closed with 3-0 silk sutures applied in an interrupted pattern. After surgery, mice were provided analgesia [intraperitoneal injection of buprenorphine (0.5 mg/kg)] every 12 hours for 72 hours and the sutures were removed after 10 days.

Animals began daily subcutaneous injection of sterile 0.9% NaCl (Braun), Forteo (40 μg/kg) that was purchased from Eli Lilly, or recombinant human PTH(1–34) (40 μg/kg) that was purchased from Sigma. The daily injections commenced immediately or 8 weeks after surgery and continued until recovery of tissues for analysis. Teriparatide dosage was chosen based on two studies demonstrating that the dose range of 30 to 40 μg/kg per day accelerates fracture healing in the mouse, primarily through enhanced chondrogenesis and expansion of the cartilaginous callus (65, 66).

Murine tissue fixation and histology preparation

A previously established systematic approach to preparation, sectioning, and visualizing articular cartilage was used for all tissue-based assays (38). At the time of harvest, mice were euthanized using an American Veterinary Medical Association (AMVA)–approved method and the surgically manipulated knee joints were dissected with the femur and tibia intact to maintain the structural integrity of the joint. Tissues were fixed at room temperature in 10% neutral buffered formalin for 72 hours, decalcified in 14% (w/v) EDTA for 14 days, processed using a microwave processor, and embedded in paraffin. Tissue blocks were then serially sectioned in the midsagittal plane through the medial compartment of the joint. A series of 3-μm-thick sections were cut at three levels within the medial compartment, each level being 50 μm from the previous level. These cut sections were mounted on positively charged glass slides, baked at 60°C overnight, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol. Some sections were stained with Alcian Blue and Orange G for OARSI scoring and histomorphometry. Unstained sections were used for immunohistochemistry and in situ hybridization.

Molecular analysis of tissues

Human and mouse sections evaluated by immunohistochemistry were treated with 3% hydrogen peroxide for 20 min, followed by a 1:20 dilution of normal goat serum for 20 min. Slides were incubated overnight at 4°C with a mouse anti-human PTHR1 monoclonal antibody (1:50; Upstate Cell Signaling), a rabbit anti-mouse JAG1 polyclonal antibody (1:100; Chemicon International), a mouse anti-human MMP13 monoclonal antibody (1:200; Thermo Scientific), a mouse anti-human COL10a1 monoclonal antibody (1:50; Quartett Immunodiagnostika & Biotechnologie), a mouse anti-human C-terminal aggrecan neoepitope NITEGE (1:400; MD Bioproducts), or a mouse anti-human RUNX2 monoclonal antibody (1:100, MBL). After this, slides were rinsed with phosphate-buffered saline (PBS) and incubated for 30 min at room temperature with a biotinylated goat anti-mouse immunoglobulin G (IgG) (1:200; Vector; for PTHR1 and COL10a1), goat anti-rabbit IgG (1:200; Vector; for JAG1), horse anti-mouse/rabbit/goat IgG (1:200; Vector; for MMP13 and NITEGE), or rabbit anti-mouse IgG (1:200; Vector; for RUNX2). Antibody binding to antigen was detected after application of horseradish peroxidase–streptavidin (1:250) with a 5-min application of Romulin AEC Chromogen (Biocare Medical). Nuclei were counterstained for 20 s with Tacha’s AEC Chromogen (Biocare Medical). In situ hybridization analysis of mouse tissues was performed as described (81, 82) using 35S-labeled RNA probes. The Col1a1 probe was developed previously (82), and the Prg4 probe was generated from a plasmid containing a complementary DNA (cDNA) clone of the gene (Open Biosystems; clone #40140700).

Mouse articular cartilage mRNA isolation and qPCR analysis

Eight weeks after surgery, mice were euthanized and tibial plateau cartilage from MLI or sham-operated joints was carefully dissected with the aid of surgical loops. Dissected cartilage was immediately frozen on dry ice and stored at −80°C until extraction of total RNA. RNA was isolated from pooled tibial plateau cartilage from 15 joints via homogenization, followed by digestion in TRIzol according to the manufacturer’s instructions. One microgram of total RNA was used to make cDNA using SuperScript II reverse transcriptase (Invitrogen). The abundance of mouse β-actin and various genes of interest was then assessed by qPCR using a Rotor Gene 6000 PCR machine (Corbett Research) using the following primer sets: 5′-TGTTACCAACTGGGACGACA-3′ and 5′-CTGGGTCATCTTTTCACGGT-3′ (β-actin); 5′-ACTCCTTCCAGGGATTTTTTGTT-3′ and 5′-GAAGTCCAATGCCAGTGTCCA-3′ (Pthr1); 5′-ATCCAGCTAAGACACAGCAAGCCA-3′ and 5′-TGGAGCACAAAGGAGTGGTCTCAA-3′ (Mmp13); and 5′-GCTACTGCACAGGGAAGAGG-3′ and 5′-TGCATATTTGGGAACCCATT-3′ (Adamts5).

Mouse OARSI scoring of cartilage

Semiquantitative histopathologic grading was performed with a derivative of the Chambers scoring system (74, 83) that has been established by the OARSI histopathology initiative as the standard method for grading of mouse cartilage degeneration (84). Based on this system, cartilage grading was carried out using Alcian Blue/Orange G–stained midsagittal sections with the following scale: 0 = normal cartilage, 0.5 = loss of proteoglycan stain without cartilage damage, 1 = mild superficial fibrillation, 2 = fibrillation and/or clefting extending below the superficial zone, 3 = mild (<25%) loss of cartilage, 4 = moderate (25 to 50%) loss of cartilage, 5 = severe (50 to 75%) loss of noncalcified cartilage, and 6 = eburnation with >75% loss of cartilage. Grading was performed by three blinded observers (R.N.R., M.J.Z., and E.R.S.). Observer agreement was evaluated in pairs via calculation of a weighted κ coefficient, using Fleiss-Cohen weights, as we have described (38). The E.R.S. versus R.N.R. coefficient was 0.94, the E.R.S. versus M.J.Z. coefficient was 0.90, and the R.N.R. versus M.J.Z. coefficient was 0.89, all indicative of strong agreement between the observers.

The three grades for each section were averaged, and the data from each group of mice were combined.

Histomorphometric determination of cartilage area

A blinded observer (E.R.S.) quantified articular cartilage area via histomorphometry, as previously described (38). Briefly, using Alcian Blue/Orange G–stained sections, the OsteoMetrics system was used to quantify articular cartilage area on one tissue section at each of three levels (50 μm apart) in the medial compartment of every joint. Using the OsteoMetrics stylus, we outlined projected images of the articular cartilage, obtained with an Olympus microscope (40× objective) outfitted with a video camera, on the femoral condyle and tibial plateau in an area that was 200 μm wide (centered on the joint). Zones of erosion and calcified cartilage were excluded from outlined area. Then, using an area-calculating algorithm in the OsteoMeasure software, we quantified the area of collected regions of interest for every section. Area values for every section from a given joint were then averaged.

Quantification of Alcian Blue staining intensity

One representative Alcian Blue/Orange G–stained section from each joint (MLI and sham) of each experimental animal was photographed and analyzed with ImageJ by splitting the color image into its corresponding red, green, and blue channels. The derivative red channel image was then inverted. A threshold of 155 to 255 was selected for all inverted images. Values that fell within this range were taken to be regions of Alcian Blue staining. The gray scales within the selected threshold range were integrated and normalized to the mean integrated intensities of articular cartilage from the saline-treated group.

μCT assessment of bone volume

Before histologic processing, harvested knee joints were evaluated via μCT using a Scanco vivaCT40 scanner with a 55–kilovolt peak (kVp) source as we have previously described (38). Joints were scanned at a resolution of 12 μm with a slice increment of 10 μm from mid-femur to mid-tibia. Images from each group were reconstructed at identical thresholds to allow three-dimensional structural rendering of each joint. Histomorphometric analysis of bone volume was performed on selected regions between the femoral and the tibial physes.

Quantification of osteophytes

Osteophyte number and diameter were assessed by a blinded observer (E.R.S.) on projected 40× images of Alcian Blue/Orange G–stained sections from each of three levels (50 μm apart) in the medial compartment of every joint. Osteophytes at both the anterior and the posterior margins were included in assessments. Using methods reported by Janusz et al. (77), we determined the mean osteophyte diameter (measured in two dimensions) using a graticule with 100-μm increments.

Statistical analyses

For all qPCR analyses, mouse OARSI scoring, quantification of Alcian Blue staining, and cartilage area determinations, statistically significant differences were identified by unpaired, two-tailed Student’s t tests, with P values <0.05 denoting significance. For μCT-based quantification of bone volume, statistically significant differences were identified by analysis of variance (ANOVA), again with P values <0.05 denoting significance. All error bars represent SEM except in the μCT analyses, where the whiskers represent the value of the maximum and minimum data point in the set.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/3/101/101ra93/DC1

Fig. S1. Immediate administration of teriparatide increases proteoglycan abundance in articular cartilage 4 weeks after MLI.

Fig. S2. Delayed PTH treatment induces chondroregeneration in joints with MLI.

Fig. S3. Teriparatide does not enhance osteophyte formation in MLI joints.

Footnotes

  • These authors contributed equally to this work.

References and Notes

  1. Acknowledgments: We thank E. Dussmann, R. Maynard, N. Miller, and N. Alcock for excellent technical assistance and M. Thullen for performing μCT analyses. We also thank C. Beck (Biostatistics Department, University of Rochester Medical Center) for advice and help with statistical analysis of raw data. For processing and sectioning of tissue samples, as well as for performing immunohistochemistry and in situ hybridization, we thank R. Tierney, S. Mack, A. Mirando, N. Porecha, and A. Thomas (Histology, Biochemistry, and Molecular Imaging Core, Center for Musculoskeletal Research). Funding: This work was supported by NIH/National Institute of Arthritis and Musculoskeletal and Skin (NIAMS) P50 AR054041 (R.N.R.), NIH/NIAMS R01 AR045700 (R.N.R.), and an Arthritis Foundation Arthritis Investigator Award (M.J.Z.). E.R.S. was supported by NIH/NIAMS T32 AR053459. Author contributions: Study design and experimental planning: E.R.S., M.J.H., R.A.M., D.C., E.M.S., S.V.B., R.J.O., H.A., J.E.P., R.N.R., and M.J.Z. Execution of experiments and data collection: E.R.S., M.J.H., Y.T., R.N.R., and M.J.Z. Data analysis and interpretation: E.R.S., M.J.H., R.A.M., E.M.S., R.J.O., H.A., J.E.P., R.N.R., and M.J.Z. Preparation of the manuscript: E.R.S., R.N.R., and M.J.Z. Competing interests: S.V.B. is a consultant for Eli Lilly; E.R.S., H.A., S.V.B., R.J.O., J.E.P., R.N.R., and M.J.Z. declare U.S. Provisional Patent Application No. 61/104,942 entitled “Protecting and repairing cartilage and musculoskeletal soft tissues” related to this work. The other authors declare that they have no competing interests.
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