How To Repair Bone Loss In Toes

Am J Pathol. 2004 Feb; 164(ii): 543–555.

Repair of Local Os Erosions and Reversal of Systemic Os Loss Upon Therapy with Anti-Tumor Necrosis Cistron in Combination with Osteoprotegerin or Parathyroid Hormone in Tumor Necrosis Factor-Mediated Arthritis

Kurt Redlich,^* Birgit Görtz,^* Silvia Hayer,^* Jochen Zwerina,^* Nicholas Doerr,^† Paul Kostenuik,^† Helga Bergmeister,^‡ George Kollias,^§ Günter Steiner,^* ^¶ Josef S. Smolen,^* ^¶ and Georg Schett^*

Kurt Redlich

From the Section of Internal Medicine III,^* Division of Rheumatology, and the Found of Biological Sciences,^‡ Academy of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Grand Oaks, California; and the Molecular Genetics Laboratory,^§ Found of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Greece

Birgit Görtz

From the Department of Internal Medicine III,^* Division of Rheumatology, and the Constitute of Biological Sciences,^‡ University of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Section of Pathology,^† Amgen, Incorporated, Chiliad Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Enquiry Heart, Vari, Greece

Silvia Hayer

From the Department of Internal Medicine III,^* Division of Rheumatology, and the Found of Biological Sciences,^‡ Academy of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Establish of Immunology, Alexander Fleming Biomedical Sciences Research Heart, Vari, Greece

Jochen Zwerina

From the Department of Internal Medicine Iii,^* Partition of Rheumatology, and the Institute of Biological Sciences,^‡ University of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian University of Sciences, Vienna, Austria; the Section of Pathology,^† Amgen, Incorporated, Chiliad Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Inquiry Center, Vari, Greece

Nicholas Doerr

From the Department of Internal Medicine III,^* Division of Rheumatology, and the Establish of Biological Sciences,^‡ University of Vienna, Vienna, Republic of austria; the Center of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Republic of austria; the Department of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Hellenic republic

Paul Kostenuik

From the Department of Internal Medicine III,^* Division of Rheumatology, and the Constitute of Biological Sciences,^‡ Academy of Vienna, Vienna, Austria; the Centre of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Grand Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Inquiry Center, Vari, Hellenic republic

Helga Bergmeister

From the Department of Internal Medicine 3,^* Sectionalization of Rheumatology, and the Institute of Biological Sciences,^‡ University of Vienna, Vienna, Austria; the Heart of Molecular Medicine,^¶ Austrian University of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Plant of Immunology, Alexander Fleming Biomedical Sciences Inquiry Center, Vari, Hellenic republic

George Kollias

From the Department of Internal Medicine 3,^* Segmentation of Rheumatology, and the Institute of Biological Sciences,^‡ University of Vienna, Vienna, Austria; the Centre of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, K Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Greece

Günter Steiner

From the Department of Internal Medicine Iii,^* Division of Rheumatology, and the Institute of Biological Sciences,^‡ University of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Constitute of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Greece

Josef S. Smolen

From the Department of Internal Medicine III,^* Partitioning of Rheumatology, and the Establish of Biological Sciences,^‡ Academy of Vienna, Vienna, Austria; the Heart of Molecular Medicine,^¶ Austrian Academy of Sciences, Vienna, Austria; the Department of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Institute of Immunology, Alexander Fleming Biomedical Sciences Enquiry Center, Vari, Greece

Georg Schett

From the Department of Internal Medicine III,^* Division of Rheumatology, and the Institute of Biological Sciences,^‡ Academy of Vienna, Vienna, Austria; the Center of Molecular Medicine,^¶ Austrian University of Sciences, Vienna, Austria; the Section of Pathology,^† Amgen, Incorporated, Thousand Oaks, California; and the Molecular Genetics Laboratory,^§ Establish of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Greece

Abstract

Local bone erosion and systemic bone loss are hallmarks of rheumatoid arthritis and crusade progressive inability. Tumor necrosis factor (TNF) is a primal mediator of arthritis and acts catabolically on os by stimulating bone resorption and inhibiting os formation. We hypothesized that the concerted action of anti-TNF, which reduces inflammation and parathyroid hormone (PTH), which stimulates os formation, or osteoprotegerin (OPG), which blocks os resorption and could pb to repair of local bone erosions and reversal of systemic bone loss. To test this, human TNF-transgenic mice with established erosive arthritis and systemic bone loss were treated with PTH, OPG, and anti-TNF, alone or in combination. Local os erosions almost fully regressed, on combined treatment with anti-TNF and PTH and/or OPG, suggesting repair of inflammatory skeletal lesions. In contrast, OPG and anti-TNF lone led to arrest of bone erosions just did not reach repair. Treatment with PTH solitary had no influence on the progression of bone erosions. Local bone erosions all showed signs of new bone germination such as the presence of osteoblasts, osteoid formation, and mineralization. Furthermore, systemic bone loss was completely reversed on combined treatment and this event was mediated by osteoblast stimulation and osteoclast blockade. In summary, we conclude that local articulation devastation and systemic inflammatory bone loss because of TNF can regress and that repair requires a combined approach by reducing inflammation, blocking os resorption, or stimulating bone formation.

Chronic inflammation and os loss are closely linked. This is impressively illustrated in rheumatoid arthritis (RA), a chronic inflammatory affliction of the joints, which is characterized past synovial inflammation and cartilage and bone destruction. Tumor necrosis cistron (TNF) is a cardinal inflammatory mediator of RA and its therapeutic inhibition leads to dramatic improvement in signs and symptoms of RA.1–four In addition, TNF has profound effects on bone: overexpression of TNF is non only involved in local bone erosion, only also induces generalized os loss.five,half dozen Therefore, TNF can be considered as an of import link between chronic inflammation and bone loss.

At a cellular level, TNF affects os past its potential to inhibit osteoblast function and its ability to stimulate osteoclast formation.7,8 The function of TNF as a stiff stimulator of osteoclasts directed attention to the part of osteoclasts in local bone erosion of arthritis. It has been shown that inflammatory os erosions in human RA and experimental arthritis contain abundant amounts of osteoclasts.9,10 Furthermore, osteoclasts play an essential role in the development of such lesions, as unequivocally demonstrated in arthritis models, which lack osteoclasts. Thus, mice defective essential mediators of osteoclastogenesis, such as c-Fos or receptor-activator of nuclear cistron-κB ligand (RANKL), develop synovial inflammation, only no bone erosions on induction of arthritis past allowed complexes or TNF overexpression, respectively.eleven,12

Therapeutic interventions to boring or abort local bone erosions are important because joint devastation is a major determinant of functional inability in human being RA.13 Therapies that inhibit os erosions target molecules involved in the differentiation and activation of osteoclasts.iii,4,xiv Inhibition of TNF and interleukin-i in human being RA past antibodies, soluble receptors, or receptor antagonists are important in currently used clinical examples,2–4,14 and experimental therapy with osteoprotegerin (OPG), a decoy receptor of RANKL, slows or even completely blocks bone erosion in animal models of arthritis.ten,15,sixteen

Although there is occasional radiological evidence for healing of erosions, it is unclear, however, whether bone erosions, in one case they have been formed, can regress and be restored by normal os.17–nineteen Such healing may require osteoblasts, which is the prison cell competent to build up new bone. Blockade of osteoclasts slows or, at best, blocks os erosion, just may not be sufficient in repair of erosions, which probable requires the synthesis and mineralization of bone matrix. Thus, repair of local bone erosions may require new approaches, such every bit combination of agents that pb to 1) reduction of the inflammatory procedure of arthritis that fuels bone erosion, 2) blockade of the resorptive chapters of arthritis past inhibiting osteoclasts, and 3) formation of new bone past stimulation of osteoblasts.

We addressed this question using human tumor necrosis factor transgenic (hTNFtg) mice that develop erosive arthritis.v Treatment was initiated long after local os erosions and systemic bone loss had already adult. Following the above hypothesis, we applied a TNF-blocker (for interference with the inflammatory response), OPG (to direct touch osteoclast differentiation and activation), and parathyroid hormone (PTH) to stimulate osteoblast action every bit single therapies or in combination.

Materials and Methods

Animals and Treatments

The heterozygous Tg197 transgenic mice have been described previously.5 Briefly, mice were fabricated transgenic for the human TNF-α gene construct with an unmodified v′promotor region, but allowing deregulated human TNF-α factor expression in vivo. Preparation of the hTNF gene constructs used has been described in item.5 These mice develop a chronic inflammatory and destructive arthritis within 5 weeks after nascence. All mice were bred and maintained in a CBAxC57BL/6 genetic background. Mice were fed a standard diet with h2o ad libitum. All animal procedures were approved by the local ethical committee.

A total number of 68 hTNFtg mice were assessed for clinical signs of arthritis (come across below) upwardly to 10 weeks after birth, ie, 5 weeks after initiation of the arthritic process. At this fourth dimension bespeak mice were matched co-ordinate to clinical signs of arthritis into vi groups every bit follows: group 1 (n = 12) was sacrificed at the beginning of handling (week 10) and served as baseline control. Group 2 (due north = 8) was left untreated up to week 14 and served every bit positive control. Grouping 3 (due north = eight) received 10 mg/kg of OPG intraperitoneally every third day. Group iv (n = 8) received 80 μg/kg of PTH 5 days per week subcutaneously. Group 5 (n = 8) received 10 mg/kg of infliximab (anti-TNF) intraperitoneally every third solar day. Groups 6, 7, and 8 (each due north = 4) received a combination of OPG+PTH, anti-TNF+OPG, and anti-TNF+PTH, respectively, applied every bit indicated above. Group 9 (due north = 12) received anti-TNF+OPG+PTH (combination). Two independent experiments were performed. All mice were littermate controlled. Treatment was applied betwixt weeks 10 and fourteen and mice were and so sacrificed. Doses of OPG, PTH, and anti-TNF were based on those successfully applied in previous studies on blocking os resorption, stimulation of bone formation, and anti-inflammatory activity, respectively.10,20,21

Reagents

Fc-OPG and PTH were kindly provided by Amgen (Thousand Oaks, CA). Fc-OPG is a truncated class of human OPG (amino acids 22 to 194) fused to the Fc domain of human IgG1 and produced in Escherichia coli.22 PTH is the one to 34 amino terminal peptide of PTH, which contains all functional backdrop of the full-length hormone. The chimeric monoclonal anti-TNF antibody (infliximab) was from Centocor (Leiden, The Netherlands).

Cell Culture

Cultures of osteoblasts and osteoclasts were performed as previously described.8,23 In brief, bone marrow was removed from femoral bones by flushing with α-minimal essential medium. For culturing osteoblasts, cells were plated at 10^iv cells/cm² and cultivated in α-minimal essential medium supplemented with 10% fetal dogie serum, 5 mmol/L glycerophosphate, and 100 μg/ml ascorbic acid. Afterwards ten days, histochemical staining of element of group i phosphatase (AP) was performed. Briefly, cells were stock-still with neutral-buffered formalin for xx minutes at 4°C, incubated with substrate solution at room temperature for 15 minutes, rinsed five times with distilled water, and photographed (F601; Nikon, Tokyo, Japan). AP-substrate was prepared from solution A (eight mg naphtol-As-TR phosphate/300 μl N,N′-dimethylformamide; both from Sigma, St. Louis, MO) and solution B (24 mg fast blue BB common salt/30 ml of 100 mmol/50 Tris-HCl, pH 9.vi). Both solutions were mixed, x mg of MgCl₂ was added, and used immediately after sterile filtration. Enzymatic activity was adamant in jail cell lysates that were solubilized with 0.1% Triton X-100. Aliquots (20 μl) of each sample were incubated with 100 μl of AP substrate buffer (100 mmol/L diethanolamine, 150 mmol/L NaCl, two mmol/L MgCl₂, p-nitrophenylphosphate at two.5 μg/ml) for v to 30 minutes at room temperature. Total cellular poly peptide was determined using the bicinchoninic method (Pierce Chemical Co., Rockford, IL). AP activity is expressed every bit U/mg protein with i U defined every bit enzymatic activity that released 1 nmol of p-nitrophenol/minute. Os nodule formation was assessed after 21 days of civilisation using alizarin crimson staining (Sigma).

For osteoclast culture, bone marrow mononuclear cells were isolated past Ficoll gradient centrifugation. Cells were plated and cultivated in α-minimal essential medium supplemented with 20 ng/ml of Yard-CSF (R&D Diagnostics, Minneapolis, MN) and 50 ng/ml RANKL (R&D). Tartrate-resistant acid phosphatase (TRAP) staining was performed subsequently 5 days of culture using a leukocyte acid phosphatase kit (Sigma).

Clinical Assessment of Arthritis

Clinical evaluation was started 6 weeks after birth and performed weekly up to week 14. Arthritis was evaluated in a blinded mode every bit described previously.11 Briefly, articulation swelling was examined using a clinical score graded from 0 to 3 (0, no swelling; i, mild-; ii, moderate-; or 3, astringent swelling of toes and ankle). In addition, grip forcefulness of each paw was analyzed on a wire of iii mm in diameter using a score from 0 to −four (0, normal grip strength; −i, mildly-; −ii, moderately-; −3, severely reduced grip strength; −4, no grip force at all). The last evaluation was performed xiv weeks after nativity. Animals were sacrificed past cervical dislocation, the blood was withdrawn by heart puncture and the paws of all animals were confused for histology.

Joint Histology

Paws were fixed in seventy% ethanol and embedded undecalcified in methylmetacrylate (M-Plast; Medim, Buseck, Germany). After polymerization sections of 3 μm were fabricated on a Jung microtome (Jung, Heidelberg, Frg), deplastinated, and stained by Goldner trichrome, von Kossa, and toluidine blue. Bone erosions were assessed at a magnification of ×200 using a Zeiss microscope (Axioskop two; Zeiss, Marburg, Germany) and quantitated past OsteoMeasure program 2.two (Osteometrics, Atlanta, GA). Numbers of osteoclasts (N.Oc) and osteoblasts (N.Ob) within inflammatory lesions were identified by their staining and morphology using histomorphometry of Goldner trichrome-stained sections. Osteoid-covered surface was also assessed using Goldner trichrome-stained sections and was related to surface area of erosions. Furthermore, the fraction of osteoid area per osteoblast (O.Ar./OB) was calculated. For dynamic histomorphometry mice were injected with calcein-3 one twenty-four hour period before sacrificing. Undecalcified hand sections were and so analyzed for fluorescence labeling at 495 nm.

Densitometry

As previously described,six bone mineral density (BMD) was quantified in ethanol-stock-still, undecalcified bones past peripheral quantitative computed tomography (pQCT) analysis (XCT-960 mol/Fifty tomograph; Norland Medical Systems, Ft. Atkinson, WI; XMICE 5.2 statistical software; Stratec, Pforzheim, Germany). BMD was measured in the tibia for the total zone (ie, the unabridged bone), the trabecular (ie, the inner twenty% of bone mass), and the cortical zone. For the total and trabecular zones, values from ii 1.25-mm sections, located 0.five mm apart, taken 1.5 mm distal to the proximal articular surface were averaged. In dissimilarity, BMD in the cortical zone of the tibia was measured in a i.25-mm section located iv.0 mm distal to the articular surface. The total and trabecular regions were differentiated from soft tissue using a separation threshold of 1500, whereas for delineating the cortical zone a threshold of 2000 was used.

Bone Histomorphometry

Left tibial bones were stock-still in 70% ethanol and embedded undecalcified in methylmetacrylate as described above and stained by Goldner trichrome. Histomorphometry was performed at a magnification of ×200 using a Zeiss microscope (Axioskop 2) and the OsteoMeasure program 2.2 (Osteometrics). The following parameters were measured according to international standards:24 fraction of bone book of total sample volume (BV/Idiot box), trabecular number (Tb.N), trabecular thickness (Tb.Th), numbers of osteoclasts per bone perimeter (N. OC/B.Pm), and numbers of osteoblasts per bone perimeter (Northward. OB/B. Pm). In addition, the width of primary spongiosa (Wi.PS) was assessed.

Serum Parameters of Bone Metabolism and hTNF Levels

Serum levels of deoxypyridinoline (DPD) crosslinks were measured by enzyme immunoassay (Quidel, San Diego, CA) after previous hydrolysis of serum samples co-ordinate to the manufacturer's recommendations. Osteocalcin was measured by immunoradiometric assay (Immutopics, San Clemente, CA). Serum levels of human being TNF were assessed by enzyme immunoassay (R&D).

Statistical Assay

Information are shown as means ± SEM. Group mean values were compared by two-tailed Student'south t-exam.

Results

hTNFtg Mice Evidence Increased Germination of Osteoclasts only Decreased Germination of Osteoblasts

To address the potential of hTNFtg mice to generate osteoclasts and osteoblasts, we investigated differentiation of bone marrow mononuclear cells into osteoclasts and of bone marrow stromal cells into osteoblasts. Formation of TRAP-positive cells on stimulation with M-CSF and RANKL was increased in hTNFtg mice compared to wild-type controls (Figure 1; a to c). Furthermore, size of osteoclasts was significantly (P < 0.01) increased in hTNFtg mice compared to wild-type controls (mean ± SEM, 0.0021 mm² ± 0.0001 versus 0.0014 mm² ± 0.00007) indicating increased osteoclastogenesis. In contrast, AP production (Effigy 1; d to f) and bone nodule formation (Figure 1; 1000 to i) by bone marrow stromal cells of hTNFtg mice was decreased, suggesting dumb osteoblast differentiation. These data not simply confirmed the basis underlying the destructive procedure namely osteoclast hyperactivity, but as well provide an explanation for the lack of repair procedure in TNF-mediated arthritis, namely reduced osteoblast activity.

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hTNFtg mice evidence increased ex vivo osteoclastogenesis only decreased osteoblast formation. Bone marrow mononuclear cells derived from wild-blazon (a) and hTNFtg (b) mice were cultured in the presence of 20 ng/ml of M-CSF and l ng/ml of RANKL for 5 days. c: TRAP staining revealed significantly increased formation of osteoclasts in hTNFtg mice compared to wild-type (wt) mice. Bone marrow stromal cells derived from wild-type (d, thousand) and hTNFtg (e, h) mice were cultivated in the presence of five mmol/50 of glycerophosphate and 100 μg/ml of ascorbic acid. AP staining (d, e), measurement of AP enzyme activeness (f), as well as Alizarin Scarlet Southward-stained os nodule germination (thousand, h, i) revealed significantly impaired formation of osteoblasts in hTNFtg mice compared to wild-type mice. Data are mean ± SEM. Asterisks bespeak significant (P < 0.01) difference. Original magnifications: ×100 (a, b, d, east); ×4 (g, h).

Joint Inflammation Is Merely Affected by Anti-TNF but Not OPG or PTH

At baseline (week 10), all hTNFtg mice had already undergone v weeks of active arthritis equally seen clinically (Figure two, a and b) and histologically (Figure 2, c and e). Moreover, local os erosions containing numerous osteoclasts were detectable (Figure 2, d and f). At this time point treatment was initiated and continued until week 14. Although hTNFtg command mice, as well equally mice treated with OPG or PTH solitary (Figure 2, a and b) or together (data not shown) progressed in mitt swelling and continued to lose grip strength, mice treated with anti-TNF showed a significantly improved course of disease (Figure 2, a and b). Anti-TNF along with OPG (data not shown) or PTH (data non shown) or OPG+PTH (Figure 2, a and b), was equally effective, suggesting that PTH and OPG, solitary or in combination, did not significantly alter the efficacy of anti-TNF on joint inflammation.

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Articulation inflammation is simply afflicted by anti-TNF but non OPG or PTH. Clinical form of arthritis indicated past joint swelling (a) and grip forcefulness (b) was assessed in hTNFtg mice between weeks 6 and 10 (pretreatment flow, black squares) and after randomization (weeks x to 14) into the post-obit handling groups: untreated (black squares), OPG (blackness triangles), PTH (white circles), anti-TNF (blackness diamonds), and OPG+PTH+anti-TNF (white squares). Asterisks bespeak a significant (P < 0.01) decrease in articulation swelling and increment in grip strength in anti-TNF-treated mice as well as in OPG+PTH+anti-TNF-treated mice compared to untreated hTNFtg controls (black squares). Pointer indicates start of treatment. hTNFtg mice develop progressive arthritis betwixt weeks 5 and 10 every bit indicated by joint swelling (a) and loss of grip strength (b). c, d, e, f: At baseline assessment (week 10) hTNFtg mice prove non only signs of synovial inflammation, just also subchondral bone erosions every bit indicated by H&E staining (c, e) and TRAP-positive multinucleated cells representing osteoclasts equally shown by TRAP-stained (d, f) histological sections of hind paws (tarsal surface area). Original magnifications: ×25 (c, d); ×100 (e, f).

Anti-TNF in Combination with OPG or PTH Leads to Repair of Bone Erosions

As illustrated before (Figure 2, d and f), histological analyses revealed os erosions in all hTNFtg mice at baseline, confirming a severely destructive disease state at the onset of treatment. The natural course of affliction showed a further increase in bone damage, as evident from mice left untreated from week x to calendar week xiv (Figure 3, a, c, and d). This is depicted in Figure 3, c and d, which reveals meaning loss of juxtaarticular bone (stained blackness by van Kossa labeling) between baseline and week 14. Treatment with PTH lonely (Figure three, a and f) did non bear upon this progression, showing a similar caste of bone erosion every bit plant in untreated mice. In contrast, OPG, anti-TNF, and OPG+PTH led to an arrest of os erosion, indicated by an amount of bone damage similar to that observed in the baseline control group (Effigy 3; a, e, and yard). However, mice treated with anti-TNF forth with OPG, PTH, or OPG+PTH, respectively, had most no bone erosions (Effigy 3, a and h). This indicated that bone erosions already present at the onset of therapy at week 10 had regressed and that, therefore, local repair processes had been induced by the therapeutic regimens.

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Repair of bone erosions on treatment with anti-TNF combined with OPG and/or PTH. a: Mean areas of local bone erosions revealed erosive disease in hTNFtg mice at baseline (week ten); progression of erosions in untreated and PTH-treated mice; and arrest in OPG, OPG+PTH, and anti-TNF-treated mice during the following four weeks. Mice treated with anti-TNF in combination with OPG, PTH, or OPG+PTH showed near complete repair of bone erosions during this period. Asterisks bespeak significant (P < 0.01) differences between treatment groups and baseline. b–h: Caste of os destruction is illustrated by von Kossa-stained histological sections (tarsal areas) of wild-type (wt) mice (b), baseline (10 weeks) hTNFtg mice (c), as well as in untreated (d), osteoprotegerin (OPG) (e), parathyroid hormone (PTH) (f), infliximab (anti-TNF) (g), and OPG+PTH+anti-TNF (combination)-treated hTNFtg mice (h). Note the fragmentation of bone (black) by synovial inflammatory tissue (scarlet). Original magnifications, ×25.

On the basis of our observations that bone erosions tin backslide, we searched for evidence of bone formation within erosions. As depicted in serial sections (Figure 4), bone erosions showed all signs of new os formation in vicinity to osteoclast-mediated bone resorption. Figure 4a shows subchondral os erosion stained with toluidine blue. The area below the articular cartilage, which normally contains subchondral bone, is filled with invasive inflammatory tissue, while articular cartilage shows signs of proteoglycan loss. Goldner-trichrome staining of the aforementioned lesion shows that this subchondral os erosion consists of two subcompartments with dissimilar bone metabolism. (Effigy 4b). Whereas i compartment (adjacent to cartilage) shows a resorption lacuna with an osteoclast, the other subcompartment shows numerous osteoblasts and osteoid formation, merely no osteoclasts. This is further illustrated by von Kossa staining of the aforementioned lesion. The area between osteoblasts and mineralized bone is filled past unmineralized osteoid (Figure 4c). In contrast, mineralized os is directly attached by inflammatory tissue at the area where osteoclasts form a resorption lacuna. Finally, as illustrated past calcein fluorescence, mineralization and thus completion of new bone formation occurs only in areas of bone erosions that are covered by osteoblasts and osteoid, but non next to osteoclast-mediated resorption lacunae (Effigy 4d). These observations suggest that eroded os maintained capacity to recover.

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Local bone erosions evidence all signs of functional os turnover. In series sections of a single subchondral os erosion (arrowheads) signs of bone resorption as well as new bone germination are detectable. a: Staining with toluidine blue reveals proteoglycan loss, indicated by destaining of the AC, articular cartilage; ST, inflammatory synovial tissue; and BO, erosion of subchondral bone. b: Goldner-trichrome staining reveals multinucleated osteoclasts (OCs) too every bit cubic mononuclear osteoblasts (OBs) at the forepart of erosions. c: Von Kossa staining shows an osteoid seam between osteoblasts and bone surface. d: Calcein labeling indicates a mineralization forepart underneath the osteoid seam. Original magnifications, ×200.

OPG Reduces Osteoclasts and PTH Increases Osteoblasts and Osteoid Formation in Local Bone Erosions

To test whether the observed therapeutic effects on bone erosions (Effigy 3) are related to changes in local os turnover, nosotros quantitatively assessed osteoclast and osteoblast numbers besides equally osteoid germination by osteoblasts inside erosions. Osteoclast numbers increased from baseline in untreated hTNFtg mice, reflecting the progression of erosion size (Figure 5a). In parallel, nevertheless, osteoblast numbers also increased, indicating skeletal responsiveness to increased os resorption (Figure 5b). The numbers of osteoclasts and osteoblasts were low in mice subjected to anti-TNF along with either OPG, PTH, or OPG+PTH, which was because of almost complete regression of bone erosions. Thus, osteoclast and osteoblast numbers were closely linked. To evaluate the potential of osteoblasts for repair of bone erosions, we quantified the amount of osteoid produced past osteoblasts within bone erosions as an indicator for new bone formation (osteoid expanse per osteoblast). Osteoid production was increased on therapy with PTH and particularly on treatment with anti-TNF+PTH or triple (combination) therapy, which suggests that osteoblast stimulation by PTH effectively leads to new bone formation inside arthritic os erosions (Figure 5c).

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OPG reduces osteoclast numbers and PTH increases osteoblast numbers and formation of osteoid in local bone erosions. Quantitative assessment of osteoclast (a) and osteoblast (b) numbers besides as osteoid germination (c) inside local bone erosions in baseline (10 weeks) hTNFtg mice, untreated, osteoprotegerin (OPG), parathyroid hormone (PTH), infliximab (anti-TNF), OPG+PTH, anti-TNF+OPG, anti-TNF+PTH, and anti-TNF+OPG+PTH (combination)-treated hTNFtg mice. Asterisks indicate a pregnant (P < 0.05) difference to untreated hTNFtg control mice. O.Ar/OB (mm² × ten^−iv) = area of osteoid per osteoblast.

Osteoclast Blockade Is Essential for Reversal of Systemic Bone Loss Mediated by TNF

Nosotros have previously reported that hTNFtg mice, similar to patients with RA, suffer from systemic bone loss.6 To address the question if the local changes described above were accompanied past systemic bone loss we evaluated the effects of the different treatment regimens on prolonged osteoporosis. TNF-occludent, when started at week ten, had no consequence on BMD, neither total (Figure 6a) nor trabecular (Figure 6b), compared to untreated hTNFtg control mice (Effigy 6, a and b). Likewise PTH alone or in combination with anti-TNF had no effect. In contrast, a significant increment of BMD was observed with OPG therapy (Figure 6; a, b, and e). All the same, there was an fifty-fifty more dramatic increment in BMD when OPG was combined anti-TNF, PTH, or anti-TNF+PTH (Figure six; a, b, and f). Gain in bone density was considering of an increase of trabecular os, whereas cortical os remained near unaffected (Figure six, b and c). These data together indicate that even longstanding inflammatory bone loss is reversible past osteoclast blockade with OPG, or more than finer, if osteoblasts are stimulated or inflammation is blocked simultaneously. Histological correlates of changes in bone density are depicted in Figure 6, d, due east, and f, showing reappearance of trabecular bone in the metaphyses of mice treated with OPG (Effigy 6e) and, virtually prominently with combination therapy (Figure 6f).

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Osteoclast occludent is essential for reversal of systemic os loss mediated by TNF. Quantitative computed tomography of hTNFtg mice showing total (a), trabecular (b), and cortical (c) BMD. hTNFtg mice of the following groups were analyzed: baseline control (week ten); untreated control (week xiv); and osteoprotegerin (OPG), parathyroid hormone (PTH), infliximab (anti-TNF), OPG+PTH, anti-TNF+OPG, anti-TNF+PTH, or anti-TNF+OPG+PTH (combination) treatment each from weeks 10 to 14. A significant increase (asterisk; P < 0.05) of total besides as trabecular BMD was observed after treatment with OPG lone or in combination with PTH, anti-TNF, or anti-TNF+PTH, whereas cortical BMD remained unchanged. Histological correlates are depicted in sections of tibial heads stained by von Kossa (d–f). Untreated hTNFtg mice show severe osteoporosis (d). Trabecular bone reappears in the metaphyses of mice receiving OPG alone (e) and, more than pronounced, afterwards combination therapy (f). Original magnifications, ×25.

Combinations of PTH, OPG, and Anti-TNF Increment Trabecular Os Mass past Reducing the Number of Osteoclasts and Stimulating Osteoblasts

We next specified the morphological basis of changes in bone density in hTNFtg mice. Bone volume increased significantly on treatment of hTNFtg mice with OPG alone or most prominently together with anti-TNF, PTH, or anti-TNF+PTH (Effigy 7a). Similar results were obtained in respect to the width of master spongiosa and trabecular number (Figure 7, b and c), whereas trabecular thickness only increased in mice treated with combination therapy (Figure 7d). Taken together these information indicate a recovering of bone microarchitecture later OPG. Changes in bone mass were accompanied by decreased numbers of osteoclasts in therapies containing OPG as well as increased osteoblast numbers in therapies, which included PTH (Effigy vii, e and f). Thus, combination handling is highly constructive because of synergy of osteoclast blockade with osteoblast stimulation.

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Combinations of PTH, OPG, and anti-TNF increase trabecular bone mass by reducing the number of osteoclasts and stimulating osteoblasts. Bone histomorphometry was performed in baseline (week 10) hTNFtg mice as well every bit in untreated-, osteoprotegerin (OPG)-, parathyroid hormone (PTH)-, infliximab (anti-TNF)-, OPG+PTH-, anti-TNF+OPG-, anti-TNF+PTH-, or anti-TNF+OPG+PTH (combination)-treated hTNFtg mice. The fraction of os volume of total sample book (BV/TV) (a), width of the primary spongiosa (PS) (b), and trabecular number (Tb.N) (c) were significantly increased in mice treated with OPG alone or along with PTH, anti-TNF, or anti-TNF+PTH. Trabecular thickness (Tb.Th) (d) was increased in the same groups except OPG alone. This was accompanied past a decreased number of osteoclasts per bone perimeter (N.OC/B.Pm) (e). In contrast, the number of osteoblasts per bone perimeter (N.OB/B. Pm) was increased in hTNFtg mice receiving PTH alone or along with anti-TNF or anti-TNF+OPG (f). Asterisks indicate pregnant (P < 0.05) divergence to untreated hTNFtg controls.

Finally, to accost whether the effects observed in densitometry and histomorphometry correlate with systemic changes of os metabolism in hTNFtg mice, we analyzed serum levels of DPD and osteocalcin. Levels of DPD, a marker for bone resorption, were significantly decreased after treatment with anti-TNF alone or in combination with PTH. Treatments containing OPG, nonetheless were fifty-fifty more constructive, indicating successful blockade of systemic osteoclast activity (Effigy 8a). Conversely, PTH, alone or in combination, significantly increased levels of osteocalcin, a mark of osteoblast activation and bone formation (Figure 8b). In contrast, serum levels of human TNF were not altered by treatments (data not shown). Thus, the effects of OPG and PTH on bone are reflected by profound changes in systemic bone metabolism. The combination of osteoclast blockade with osteoblast stimulation has a positive net consequence on os mineralization, in addition to that on local bone erosions.

An external file that holds a picture, illustration, etc. Object name is zjh0020439690008.jpg

Decrease of systemic os resorption by OPG and increase of systemic bone formation past PTH. Serum levels of DPD (a) and osteocalcin (OC) (b) were measured in baseline (x weeks) hTNFtg mice, every bit well as in untreated, osteoprotegerin (OPG)-, parathyroid hormone (PTH)-, infliximab (anti-TNF)-, OPG+PTH-, anti-TNF+OPG-, anti-TNF+PTH-, or anti-TNF+OPG+PTH (combination)-treated hTNFtg mice. DPD levels, a marking of os resorption well-nigh prominently decreased after treatments containing OPG, indicating effective systemic blockade of osteoclast activity. Conversely, treatments containing PTH increased OC levels, a marking of osteoblast activation and bone formation. Asterisks indicate a pregnant (P < 0.05) difference to untreated hTNFtg control mice.

Discussion

Local and generalized os loss correspond a serious trouble for patients with RA. Local bone erosion is a authentication of RA patients and is considered every bit irreversible damage associated with increased morbidity and loss of joint function.13 Generalized os loss in RA patients is a consequence of high inflammatory disease action, functional damage, and corticosteroid apply, and leads to an increased gamble of osteoporotic fracture.25,26 Repair of bone impairment, rather than slowing or at best blocking structural damage should therefore exist an ambitious aim of anti-rheumatic therapy. Previous data from in vivo experiments and clinical studies have shown that pure blockade of TNF is non sufficient to repair or even just arrest local and systemic inflammatory os loss,two–4,half dozen,ten,21 suggesting that a more profound intervention in the bone remodeling process is necessary to achieve this ambitious goal. This written report demonstrates that therapeutic regulation of bone metabolism, as achieved with the combination of PTH or OPG, dramatically improves structural joint impairment when combined with an amanuensis that controls the chronic inflammatory process, such every bit anti-TNF antibody. Moreover, combination therapy also led to a dramatic increase of systemic os mass in mice suffering from longstanding inflammatory disease and severe osteoporosis, ultimately reversing it to the physiological levels of healthy mice.

The rationale for combining TNF-blockade with osteoclast inhibition or osteoblast stimulation to overcome TNF-mediated bone harm comes from in vitro observations on the effects of TNF on osteoclasts and osteoblasts.vii TNF is a potent stimulator of osteoclast differentiation and activation, which is illustrated by increased formation of osteoclasts from mononuclear precursors.27 The consequence is mediated past induction of RANKL expression and a synergistic consequence with RANKL on nuclear factor-κB and stress-activated/c-Jun NH(ii)-terminal protein kinase, ii signaling pathways essential for osteoclastogenesis.viii With respect to the osteoblast lineage, however, TNF is considered a negative regulator that inhibits osteoblast differentiation and function. Decreased in vitro differentiation of bone marrow stromal cells into osteoblasts supports this idea.vii,28 Several mechanisms have been described, which explain the regulatory effect of TNF on osteoblasts. TNF inhibits IGF-1 and the transcription gene Runx-2, which are involved in the differentiation process of osteoblasts.29 In add-on, TNF inhibits the synthesis of matrix proteins, such as osteocalcin30 and AP,31,32 which are considered to exist indicators for osteoblast function. This is illustrated by TNF-transgenic mice, which show a negative net effect of TNF on bone resulting in local bone erosions and generalized bone loss.5,6 Thus, a combined intervention, which slows bone resorption and induces os formation, seems to be a highly constructive strategy to achieve repair of TNF-induced bone damage.

OPG can be considered to exist a feasible means of blocking TNF-mediated bone resorption, because it interferes with RANKL, which is essential for TNF-mediated osteoclastogenesis.33 OPG, given in pharmacological doses, effectively blocks bone resorption and increases systemic bone mass.34 This was considering of a predominant effect of OPG on trabecular bone, which exhibits a more active turnover than cortical bone.6 OPG also slows local bone erosion in arthritic joints, indicating that TNF and RANKL induce formation of osteoclasts in the synovial membrane, which ultimately erode subchondral bone.x,15,35 Indeed, the fact that in our model OPG treatment results in a positive net effect on bone, suggests that the primary effect of OPG is an effective occludent of os resorption. However, an inhibitory event of OPG on osteoblasts also could be observed, attributing to recent experimental findings, which identified RANKL every bit stimulator of osteoblasts and supporting observations of blunted bone formation after OPG treatment in laboratory animals and humans.6,34,36 In contrast, PTH primarily stimulates osteoblasts and its effects on osteoclasts are of indirect nature.37 PTH, when given cyclically, stimulates bone formation and leads to increased os mass.38 Considering TNF inhibits PTH-mediated effects on osteoblasts,39 therapeutic substitution of PTH seems to be a feasible strategy to stimulate os germination in the presence of TNF.

Most chiefly, local os erosions regressed after TNF blockade combined with osteoclast inhibition or osteoblast stimulation. Local erosions are mediated past osteoclasts derived from mononuclear forerunner cells within the inflamed synovial tissue. Electric current therapies for RA, such as blockade of TNF tin slow or, at best, arrest progression of local bone erosion.ii,three,40 Experimental blockade of osteoclasts by OPG or bisphosphonates also slows this process, simply does not induce the repair of such lesions. So far in that location has been no disarming show of repair mechanisms occurring in local bone erosions.ten,15,35 Recently, cells expressing PTH receptor have been described within os erosions and these cells are fastened to bone at the erosion front end.41 Because of their localization these cells well-nigh probable represent osteoblasts, although PTH receptors are expressed by synovial lining cells and fibroblast-like cells within the synovial pannus likewise.42 In this study we demonstrate that local bone erosions are an area of dynamic bone turnover. Apart from resorption lacunae formed by osteoclasts, all signs of bone formation are found inside such lesions. These include one) cells with the typical morphological characteristics of osteoblasts localized between erosion front end and bone, 2) osteoid formation adjacent to osteoblast at the interface of pannus and os, and 3) mineralization of newly formed bone. Although at that place is the potential of repair mechanisms within local bone erosion, os formation can plain not completely compensate the erosive procedure. This may take ii reasons: firstly, factors synthesized past synovial inflammatory tissue such as TNF may hamper osteoblast activity and, secondly, excessive osteoclast activity may forestall the recruitment of osteoblasts and thus remodeling of resorption lacunae. Thus, a combined therapeutic approach, which reduces the inflammatory activity of synovial tissue and blocks either osteoclast activeness or stimulates bone formation, is necessary to repair local os erosions in vivo.

Whether these findings of hTNFtg mice are too valid for human RA remains to be determined. Strengths of this model are 1) its chronic progressive grade leading to a high burden of illness, ii) a well-defined pathomechanism based on cytokine overexpression, and 3) an excellent possibility to study the local and systemic skeletal effects of inflammation. Although hTNFtg mice represent an attractive model for human RA its limitations should exist considered: specially, the model is not an autoimmune driven model of arthritis and hence does not depend on autoantibodies and T cells. Because human being RA exhibits autoimmune features, similar studies in autoimmune arthritis models may thus allow gaining further insight in the mechanisms of bone repair during inflammation. Nevertheless, these data not only describe repair mechanisms of inflammatory bone damage but also highlight differences betwixt local and systemic os loss. Thus, local os erosion results from a tight interplay between inflammation and unfavorable bone-remodeling processes. Reversal is just possible if these 2 mechanisms are targeted simultaneously. In fact, both of them appear every bit of import considering neither pure blockade of inflammation nor even complex interference with bone remodeling is sufficient to achieve regression of local os erosion. In dissimilarity, systemic bone loss is much more a result of osteoclast-mediated bone resorption, which is supported past the fact that OPG per se increases systemic os mass and by the high efficacy of all OPG-containing combination therapies.

In summary, our data show that joint destruction and generalized inflammatory bone loss are reversible. Os repair requires therapeutic intervention, which not just controls inflammation but likewise shifts the balance of osteoclasts to osteoblasts in favor of the latter. These findings open new perspectives in the handling of RA by demonstrating that bone impairment is of reversible nature rather than end-stage damage associated with loss of joint part.

Footnotes

Accost reprint requests to Georg Schett, M.D., Department of Internal Medicine III, Division of Rheumatology, Academy of Vienna, Währinger Gürtel eighteen-twenty, A-1090 Vienna, Austria. .ta.ca.neiw-hka@ttehcs.groeg :liam-Eastward

Supported by the Austrian Science Fund (START prize to G.Due south.); the Heart of Molecular Medicine of the Austrian Federal Ministry building for Teaching, Scientific discipline, and Civilisation and the City of Vienna; and the Austrian National Bank (project 8715 to One thousand.Due south.).

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