Understanding Coupling between Bone Resorption and Formation
2013; Elsevier BV; Volume: 183; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2013.03.006
ISSN1525-2191
AutoresThomas Levin Andersen, Mohamed Essameldin Abdelgawad, Helene Kristensen, Ellen‐Margrethe Hauge, Lars Rolighed, Jens Bollerslev, Per Kjærsgaard‐Andersen, Jean‐Marie Delaissé,
Tópico(s)Bone health and treatments
ResumoBone remodeling requires bone resorption by osteoclasts, bone formation by osteoblasts, and a poorly investigated reversal phase coupling resorption to formation. Likely players of the reversal phase are the cells recruited into the lacunae vacated by the osteoclasts and presumably preparing these lacunae for bone formation. These cells, called herein reversal cells, cover >80% of the eroded surfaces, but their nature is not identified, and it is not known whether malfunction of these cells may contribute to bone loss in diseases such as postmenopausal osteoporosis. Herein, we combined histomorphometry and IHC on human iliac biopsy specimens, and showed that reversal cells are immunoreactive for factors typically expressed by osteoblasts, but not for monocytic markers. Furthermore, a subpopulation of reversal cells showed several distinctive characteristics suggestive of an arrested physiological status. Their prevalence correlated with decreased trabecular bone volume and osteoid and osteoblast surfaces in postmenopausal osteoporosis. They were, however, virtually absent in primary hyperparathyroidism, in which the transition between bone resorption and formation occurs optimally. Collectively, our observations suggest that arrested reversal cells reflect aborted remodeling cycles that did not progress to the bone formation step. We, therefore, propose that bone loss in postmenopausal osteoporosis does not only result from a failure of the bone formation step, as commonly believed, but also from a failure at the reversal step. Bone remodeling requires bone resorption by osteoclasts, bone formation by osteoblasts, and a poorly investigated reversal phase coupling resorption to formation. Likely players of the reversal phase are the cells recruited into the lacunae vacated by the osteoclasts and presumably preparing these lacunae for bone formation. These cells, called herein reversal cells, cover >80% of the eroded surfaces, but their nature is not identified, and it is not known whether malfunction of these cells may contribute to bone loss in diseases such as postmenopausal osteoporosis. Herein, we combined histomorphometry and IHC on human iliac biopsy specimens, and showed that reversal cells are immunoreactive for factors typically expressed by osteoblasts, but not for monocytic markers. Furthermore, a subpopulation of reversal cells showed several distinctive characteristics suggestive of an arrested physiological status. Their prevalence correlated with decreased trabecular bone volume and osteoid and osteoblast surfaces in postmenopausal osteoporosis. They were, however, virtually absent in primary hyperparathyroidism, in which the transition between bone resorption and formation occurs optimally. Collectively, our observations suggest that arrested reversal cells reflect aborted remodeling cycles that did not progress to the bone formation step. We, therefore, propose that bone loss in postmenopausal osteoporosis does not only result from a failure of the bone formation step, as commonly believed, but also from a failure at the reversal step. Under normal physiological conditions, the strength of adult bone is maintained through constant renewal of bone matrix. This process, called bone remodeling, involves two highly coordinated events: resorption of existing bone by osteoclasts (OCs), followed by restitution of new bone by osteoblasts (OBs). In osteoporosis, bone formation becomes insufficient to reconstruct the bone matrix resorbed by the OCs, therefore leading to a reduction in bone mass and predisposing bone to fracture. This negative balance is commonly ascribed to alterations of the OC and OB activities.1Eriksen E.F. Normal and pathological remodeling of human trabecular bone: three-dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease.Endocr Rev. 1986; 7: 379-408Crossref PubMed Scopus (479) Google Scholar, 2Parfitt A.M. The coupling of bone formation to bone resorption: a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis.Metab Bone Dis Relat Res. 1982; 4: 1-6Abstract Full Text PDF PubMed Scopus (293) Google Scholar However, despite increasing knowledge on OCs and OBs, the actual mechanism securing the restitution of resorbed bone remains unknown, and none of the current clinical treatments appears to target the mechanism ensuring that OBs form bone matrix after osteoclastic bone resorption. The efforts to understand the bone remodeling mechanism have so far paid only little attention on the intermediate phase of bone remodeling known as the reversal phase, although it is, by nature, the one coupling bone resorption to bone reconstruction.3Parfitt A.M. The cellular basis of bone remodeling: the quantum concept reexamined in light of recent advances in the cell biology of bone.Calcif Tissue Int. 1984; 36: S37-S45Crossref PubMed Scopus (327) Google Scholar, 4Baron R. Vignery A. Tran Van P.T. The significance of lacunar erosion without osteoclasts: studies on the reversal phase of the remodeling sequence.Metab Bone Dis Rel Res. 1980; 2: 35-40Google Scholar In fact, bone surfaces (BSs) undergoing remodeling show not only OCs and OBs, but also mononucleated cells that colonize eroded surfaces (ESs) after the departure of the OCs. They represent as much as 80% of the ES and 25% of the remodeling surfaces of human trabecular bone.5Baron R. Vignery A. Lang R. Reversal phase and osteopenia: defective coupling of resorption to formation in the pathogenesis of osteoporosis.in: Deluca H.F. Frost H.M. Jee W.S.S. Johnston C.C. Parfitt A.M. Osteoporosis: Recent Advances in Pathogenesis and Treatment. University Park Press, Baltimore1980: 311-320Google Scholar, 6Baron R. Magee S. Silverglate A. Broadus A. Lang R. Estimation of trabecular bone resorption by histomorphometry: evidence for a prolonged reversal phase with normal resorption in post-menopausal osteoporosis and coupled increase in primary hyperparathyroidism.Clin Disorders Bone Miner Metab. 1983; : 191-195Google Scholar A systematic analysis of the distribution of these cells, previously defined as reversal cells (Rv.Cs),7Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. Bone histomorphometry: standardization of nomenclature, symbols, and units: report of the ASBMR Histomorphometry Nomenclature Committee.J Bone Miner Res. 1987; 2: 595-610Crossref PubMed Scopus (4914) Google Scholar, 8Raggatt L.J. Partridge N.C. Cellular and molecular mechanisms of bone remodeling.J Biol Chem. 2010; 285: 25103-25108Crossref PubMed Scopus (835) Google Scholar, 9Raisz L.G. Pathogenesis of osteoporosis: concepts, conflicts, and prospects.J Clin Invest. 2005; 115: 3318-3325Crossref PubMed Scopus (1290) Google Scholar revealed that they are positioned between the OCs and the OBs in human trabecular bone.10Eriksen E.F. Gundersen H.J. Melsen F. Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition.Metab Bone Dis Relat Res. 1984; 5: 243-252Abstract Full Text PDF PubMed Scopus (148) Google Scholar In a rat model in which a wave of remodeling was induced in the mandible by extraction of the opposing row of teeth, these Rv.Cs appeared after the OCs and before the OBs.11Tran Van P.T. Vignery A. Baron R. Cellular kinetics of the bone remodeling sequence in the rat.Anat Rec. 1982; 202: 445-451Crossref PubMed Scopus (123) Google Scholar, 12Tran V.P. Vignery A. Baron R. An electron-microscopic study of the bone-remodeling sequence in the rat.Cell Tissue Res. 1982; 225: 283-292Crossref PubMed Scopus (90) Google Scholar The unique position of Rv.Cs relative to OCs and OBs makes them the natural intermediates for mediating the interactions between OCs and OBs, as well as the privileged candidates for switching the molecular properties of the BS from resorbing to the opposite one reconstructing bone matrix. In this respect, it has been proposed that the early tasks of Rv.Cs are to clean the Howship's lacunae and to contribute to the generation of cement lines, thereby rendering the BS propitious to bone formation.4Baron R. Vignery A. Tran Van P.T. The significance of lacunar erosion without osteoclasts: studies on the reversal phase of the remodeling sequence.Metab Bone Dis Rel Res. 1980; 2: 35-40Google Scholar, 13Domon T. Suzuki R. Takata K. Yamazaki Y. Takahashi S. Yamamoto T. Wakita M. The nature and function of mononuclear cells on the resorbed surfaces of bone in the reversal phase during remodeling.Ann Anat. 2001; 183: 103-110Crossref PubMed Scopus (15) Google Scholar, 14Everts V. Delaisse J.M. Korper W. Jansen D.C. Tigchelaar-Gutter W. Saftig P. Beertsen W. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.J Bone Miner Res. 2002; 17: 77-90Crossref PubMed Scopus (275) Google Scholar, 15McKee M.D. Nanci A. Osteopontin and the bone remodeling sequence: colloidal-gold immunocytochemistry of an interfacial extracellular matrix protein.Ann N Y Acad Sci. 1995; 760: 177-189Crossref PubMed Scopus (98) Google Scholar, 16Mulari M.T. Qu Q. Harkonen P.L. Vaananen H.K. Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro.Calcif Tissue Int. 2004; 75: 253-261Crossref PubMed Scopus (76) Google Scholar, 17Zhou H. Chernecky R. Davies J.E. Deposition of cement at reversal lines in rat femoral bone.J Bone Miner Res. 1994; 9: 367-374Crossref PubMed Scopus (50) Google Scholar Rv.C activities were found permissive for subsequent bone formation14Everts V. Delaisse J.M. Korper W. Jansen D.C. Tigchelaar-Gutter W. Saftig P. Beertsen W. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.J Bone Miner Res. 2002; 17: 77-90Crossref PubMed Scopus (275) Google Scholar and, thus, Rv.Cs may represent the missing link necessary to understand coupling between bone resorption and formation, and to prevent osteoporosis. Surprisingly, the hypothesis that Rv.Cs may be critical for this coupling has received little attention. We do not even know the nature of the Rv.Cs, and different cell origins have been proposed: they may represent mononucleated OCs,10Eriksen E.F. Gundersen H.J. Melsen F. Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition.Metab Bone Dis Relat Res. 1984; 5: 243-252Abstract Full Text PDF PubMed Scopus (148) Google Scholar phagocytotic cells,11Tran Van P.T. Vignery A. Baron R. Cellular kinetics of the bone remodeling sequence in the rat.Anat Rec. 1982; 202: 445-451Crossref PubMed Scopus (123) Google Scholar, 12Tran V.P. Vignery A. Baron R. An electron-microscopic study of the bone-remodeling sequence in the rat.Cell Tissue Res. 1982; 225: 283-292Crossref PubMed Scopus (90) Google Scholar or OB lineage cells (ie, pre-OB cells).14Everts V. Delaisse J.M. Korper W. Jansen D.C. Tigchelaar-Gutter W. Saftig P. Beertsen W. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.J Bone Miner Res. 2002; 17: 77-90Crossref PubMed Scopus (275) Google Scholar, 16Mulari M.T. Qu Q. Harkonen P.L. Vaananen H.K. Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro.Calcif Tissue Int. 2004; 75: 253-261Crossref PubMed Scopus (76) Google Scholar Furthermore, we do not know whether malfunction of Rv.Cs may contribute to bone loss in diseases such as osteoporosis. The present study is part of an effort to investigate the hypothesis that Rv.Cs may play a role in coupling bone resorption and formation. More specifically, it addresses the question of the cellular origin of Rv.Cs and of their role in bone loss. Therefore, we performed combined histomorphometry and immunohistochemistry (IHC) on biopsy specimens obtained from patients with postmenopausal osteoporosis (PMO), corresponding postmenopausal controls (PMCs), and patients with primary hyperparathyroidism (PHPT). These biopsy specimens represent distinct situations with respect to the resorption-formation balance and, therefore, offer the possibility to investigate the relation between the resorption-formation balance and changes in the behavior of Rv.Cs. The histomorphometric analysis was conducted on plastic-embedded transiliac bone biopsy specimens (7 mm diameter) taken from 23 patients with a PMO fracture (aged 70 ± 5 years) and 10 PMCs (aged 65 ± 9 years). Three of these PMO specimens were also used for three-dimensional (3D) analysis. Plastic-embedded transiliac bone biopsy specimens (7 mm diameter) taken during the parathyroid surgery of 10 PHPT patients (eight women and two men, aged 57 ± 7 years) were also included as an additional control arm for histomorphometry; PHPT offers a situation in which coupling between bone resorption and formation occurs optimally,18Eriksen E.F. Primary hyperparathyroidism: lessons from bone histomorphometry.J Bone Miner Res. 2002; 17: N95-N97PubMed Google Scholar and was previously used as a control arm to demonstrate a prolonged/aborted reversal phase in PMO.6Baron R. Magee S. Silverglate A. Broadus A. Lang R. Estimation of trabecular bone resorption by histomorphometry: evidence for a prolonged reversal phase with normal resorption in post-menopausal osteoporosis and coupled increase in primary hyperparathyroidism.Clin Disorders Bone Miner Metab. 1983; : 191-195Google Scholar The diagnosis of the PHPT patients was based on elevated parathyroid hormone and calcium levels, according to the international guidelines.19Silverberg S.J. Lewiecki E.M. Mosekilde L. Peacock M. Rubin M.R. Presentation of asymptomatic primary hyperparathyroidism: proceedings of the third international workshop.J Clin Endocrinol Metab. 2009; 94: 351-365Crossref PubMed Scopus (275) Google Scholar The IHC characterization of the Rv.Cs was conducted on decalcified, paraffin-embedded, transiliac bone biopsy specimens (7 mm diameter) from 10 PHPT patients and iliac bone marrow core biopsy specimens (3 mm diameter) from 10 control patients (six women and four men, aged 60 ± 8 years) under investigation for a hematological malignancy, with no apparent pathological feature in the bone, who had not been subjected to any treatment known to affect bone.20Kristensen H.B. Andersen T.L. Marcussen N. Rolighed L. Delaisse J.M. Increased presence of capillaries next to remodeling sites in adult human cancellous bone.J Bone Miner Res. 2013; 28: 574-585Crossref PubMed Scopus (73) Google Scholar Bone specimens from the femur head of six PMO women undergoing hip replacement were also included in the IHC analysis and in some of the 3D analyses. The study was approved by the Danish National Committee on Biomedical Research Ethics (project S-20070121). Masson's trichrome–stained plastic sections were subjected to conventional bone histomorphometry assessing the trabecular bone volume/total volume (BV/TV) and classic BS parameters, such as the extent of ES, osteoid surface (OS/BS), osteoclast surface (Oc.S/BS), osteoblast surface (Ob.S/BS), mineralizing surface (MS), and reversal surface (Rv.S/BS), identified as ES without OCs. Wall thickness (W.Th), osteoid thickness (O.Th), mineral apposition rate (MAR), and erosion depth (E.De) were uniformly and randomly sampled along the surface. The E.De was measured by counting the number of eroded lamellae, as described by Eriksen et al.10Eriksen E.F. Gundersen H.J. Melsen F. Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition.Metab Bone Dis Relat Res. 1984; 5: 243-252Abstract Full Text PDF PubMed Scopus (148) Google Scholar W.Th, O.Th, and MAR were measured by orthogonal intercepts, according to Steiniche et al.21Steiniche T. Eriksen E.F. Kudsk H. Mosekilde L. Melsen F. Reconstruction of the formative site in trabecular bone by a new, quick, and easy method.Bone. 1992; 13: 147-152Abstract Full Text PDF PubMed Scopus (35) Google Scholar In addition to the classic histomorphometric parameters, we conducted a more specific analysis of the Rv.S. Herein, the Rv.S was divided into active Rv.S, which was directly flanked by Oc.S or OS, or arrested Rv.S without any neighboring Oc.S or OS (see Results). The number of nuclear profiles of mononuclear cells on Rv.S and quiescent surface per mm BS was measured using the Osteomeasure System (Osteometrics, Decatur, GA). The immunostaining for osteoblastic markers [Runx2, osterix, smooth muscle actin (SMA), alkaline phosphatase, and CD56] was conducted in combination with staining for the OC marker, tartrate-resistant acid phosphatase (TRAcP), whereas the immunostaining for monocytic markers (CD68, CD14, and CD163) was conducted in combination with Runx2 immunostaining, according to previously described procedures.22Andersen T.L. Sondergaard T.E. Skorzynska K.E. Dagnaes-Hansen F. Plesner T.L. Hauge E.M. Plesner T. Delaisse J.M. A physical mechanism for coupling bone resorption and formation in adult human bone.Am J Pathol. 2009; 174: 239-247Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar More precisely, the immunostaining with mouse anti-CD68 (clone KP-1; Dako, Glostrup, Denmark), anti-CD163 (clone 10D6; Novocastra, Leica Microsystems, Ballerup, Denmark), and anti-CD14 (clone 7; Novocastra) was labeled with alkaline phosphatase–conjugated anti-mouse IgG Powervision (ImmunoVision, Springdale, AR). The immunostaining for monocytic markers was combined with staining with anti-Runx2 (ab81357; Abcam, Cambridge, UK) antibodies that were labeled with horseradish peroxidase–conjugated anti-rabbit IgG EnVision (Dako). The consistency of the signals shown by the latter anti-Runx2 antibody was validated with another two monoclonal antibodies against Runx2 [clone 27-K (Santa Cruz Biotechnology, Santa Cruz, CA); and clone 1D8 (Abcam)]. The immunostaining with mouse IgG1 anti-CD56 (clone 56C05; Lab Vision, Thermo Fischer Scientific, Kalamazoo, MI) and IgG2a anti-SMA (clone 1A4; Dako) antibodies was labeled with horseradish peroxidase–conjugated anti-mouse IgG1 or IgG2a subtype-specific secondary antibodies (Jackson ImmunoResearch, Suffolk, UK), whereas immunostaining with rabbit anti-osterix (ab22552; Abcam), anti-alkaline phosphatase (ab75699; Abcam), or anti-Runx2 (ab81357; Abcam) antibodies was labeled with horseradish peroxidase–conjugated anti-rabbit IgG EnVision. The immunostaining for osteoblastic markers was combined with staining with mouse IgG2b anti-TRAcP (clone ZY-905; Invitrogen, Life Technology, Nærum, Denmark), which was labeled with alkaline phosphatase–conjugated IgG2b subtype-specific secondary antibodies. The horseradish peroxidase and alkaline phosphatase were visualized with diaminobenzidine (DAB+; Dako) and Liquid Permanent Red (Dako) and counterstained with Mayer's hematoxylin. The IHC identification of the nature of the Rv.Cs first focused on the proportion of the immunoreactivity of CD14, CD68, CD163, Runx2, alkaline phosphatase, and CD56 in Rv.Cs adjacent to OC on the ES. This characterization was based on at least two immunostained sections from each of the biopsy specimens from the 10 PHPT and six PMO patients. Also, the proportion and intensity of the nuclear immunoreactivity of the transcription factors, osterix and Runx2, and the cellular immunoreactivity of SMA were semiquantitatively analyzed in different subpopulations of Rv.Cs in control iliac bone biopsy specimens. The subpopulations that were compared are the Rv.Cs next to OCs (early Rv.Cs) versus the Rv.Cs next to OSs (late Rv.Cs) and Rv.Cs on active Rv.S versus Rv.Cs on arrested Rv.S. Semiquantitative grading was performed for each subpopulation, using the H-score (range, 0 to 200), according to the previously published equation23Detre S. Saclani J.G. Dowsett M. A “quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas.J Clin Pathol. 1995; 48: 876-878Crossref PubMed Scopus (659) Google Scholar: H-Score = ∑Pi*i, where i is the intensity of staining visually graded (absent, 0; weak, 1; and moderate-strong, 2) and Pi is the percentage of cells stained with each intensity (0% to 100%). The microscopic analysis was conducted on an upright DM2500 microscope (Leica, Wetzlar, Germany), and micrographs were obtained with a DP71 digital camera (Olympus, Center Valley, PA). The final figures were assembled using CorelDraw version 15 (Corel Corporation, Ottawa, ON, Canada). The 3D analyses were based on 50 consecutive Masson trichrome–stained sections from paraffin-embedded biopsy specimens of the femur head and neck from three PMO patients, and from plastic-embedded iliac crest biopsy specimens from PMO patients. Four stacks of consecutive sections were obtained and used to make five stacks of micrographs and to analyze 3D coherence between Oc.S, OS, and arrested and active Rv.Ss. Each stack of micrographs included two to three remodeling events. In total, five remodeling events with arrested Rv.S and seven with active Rv.S were analyzed. Two of these stacks of micrographs were converted into 3D reconstructions using Amira software version 4.0 (Mercury Computer Systems, Merignac, France), as previously described.22Andersen T.L. Sondergaard T.E. Skorzynska K.E. Dagnaes-Hansen F. Plesner T.L. Hauge E.M. Plesner T. Delaisse J.M. A physical mechanism for coupling bone resorption and formation in adult human bone.Am J Pathol. 2009; 174: 239-247Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar The parameters measured in PMO patients were compared with the same parameters measured in PMC and PHPT patients using an unpaired Student's t-test when the data were distributed in a gaussian manner; otherwise, the Mann-Whitney rank-sum test or the Kruskal-Wallis test, followed by a Dunn's multiple-comparison test, was used. The paired Student's t-test was used to compare gaussian-distributed differences between two measurements within the same subject; otherwise, the Friedman test was used, followed by a Dunn's multiple-comparison test. The gaussian distribution of the data was assessed using a D'Agostino and Pearson Omnibus normality test. The relationship between two parameters within the same group was shown in two-way scatterplots, in which the fitted lines represent the linear relationship between the two parameters. The relationship was statistically compared using Spearman's rank correlation test (rs). P < 0.05 was defined as statistically significant. All statistical analyses and graphical illustrations were performed using GraphPad Prism version 4. Our first objective was to define Rv.Cs with respect to their histological localization, morphological characteristics, and immunoreactive characteristics. Because Rv.Cs are expected to be involved in connecting resorption by OCs to bone formation by OBs, it appeared of interest to characterize these cells in situations in which the transition from resorption to formation appears to occur optimally.1Eriksen E.F. Normal and pathological remodeling of human trabecular bone: three-dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease.Endocr Rev. 1986; 7: 379-408Crossref PubMed Scopus (479) Google Scholar Biopsy specimens from PHPT patients were included for this reason. Figure 1, A and B, illustrates our definition of Rv.Ss as BSs without OCs and of Rv.Cs as the mononuclear cells covering these surfaces. The morphological characteristics of these Rv.Cs differ, dependent on their proximity to OCs and OSs, respectively. In general, Rv.Cs appear as flat cells lining the bone and with elongated nuclei, but as they get closer to the OS, they sometimes gradually become more cuboidal, resembling cuboidal collagen-depositing OBs (Figure 1B). We first paid special attention on identifying the nature of the Rv.Cs colonizing the ES right after osteoclastic resorption. An assessment based on the sections of all 10 PHPT patients showed that the Rv.Cs next to OCs were negative for monocytic markers, such as CD14, CD68, and CD163, and for the OC marker, TRAcP (Figure 1, C–H). On the BS, CD68 (Figure 1E) and TRAcP (Figure 1, D, F, and H) immunoreactivity was restricted to OCs. On the other hand, almost all Rv.Cs lining the eroded BS next to OCs showed immunoreactivity for osteoblastic markers, such as Runx2 (97%; 193 of 198 Rv.Cs) and alkaline phosphatase (88%; 118 of 134 Rv.Cs), and 75% (70 of 93) of these Rv.Cs were positive for CD56, a protein transiently expressed during OB differentiation24Lee Y.S. Chuong C.M. Adhesion molecules in skeletogenesis, I: transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification.J Bone Miner Res. 1992; 7: 1435-1446Crossref PubMed Scopus (50) Google Scholar (Figure 1, C–E and G). Next, we examined whether Rv.Cs next to osteoid represent more mature OBs compared with Rv.Cs next to OCs. We used a cohort of control patients for this analysis. We found that both of these Rv.C subpopulations equally express Runx2, an early marker of OB differentiation25Katagiri T. Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation.Oral Dis. 2002; 8: 147-159Crossref PubMed Scopus (467) Google Scholar, 26Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2827) Google Scholar (Table 1). However, osterix, which is a later marker,26Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2827) Google Scholar is expressed significantly more intensively and in more Rv.Cs next to OSs, compared with Rv.Cs next to OCs (Figure 2, C and D, and Table 1). In contrast, SMA, a stage-specific marker of osteoprogenitors,20Kristensen H.B. Andersen T.L. Marcussen N. Rolighed L. Delaisse J.M. Increased presence of capillaries next to remodeling sites in adult human cancellous bone.J Bone Miner Res. 2013; 28: 574-585Crossref PubMed Scopus (73) Google Scholar, 27Kalajzic Z. Li H. Wang L.P. Jiang X. Lamothe K. Adams D.J. Aguila H.L. Rowe D.W. Kalajzic I. Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population.Bone. 2008; 43: 501-510Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar behaves in the opposite way (Figure 2, F and G, and Table 1). Collectively, the immunostaining data indicate that the Rv.Cs colonizing resorption lacunae right after the departure of the OC are osteoblast lineage cells that mature into bone-forming osteoblasts during the reversal phase.Table 1Semiquantitative Analysis of the Immunoreactivity of Osterix, Runx2, and SMA in Rv.Cs Proximal to OC or OSVariableRv.Cs next to OCRv.Cs next to OSDifferenceP value∗The differences between Rv.C populations are compared by paired Student's t-test.OsterixNo. of Rv.Cs122151Percent stained Weak29 ± 1335 ± 195.8 ± 19NS Moderate/strong8.1 ± 1124 ± 2416 ± 250.08 Total37 ± 2359 ± 2022 ± 29<0.05H-score45 ± 3483 ± 3937 ± 51<0.05Runx2No. of Rv.Cs10870Percent stained Weak43 ± 1338 ± 22−4.1 ± 22NS Moderate/strong41 ± 2651 ± 329.4 ± 26NS Total84 ± 1988 ± 195.3 ± 19NSH-score124 ± 43139 ± 4815 ± 40NSSMANo. of Rv.Cs9473Percent stained Weak44 ± 298.3 ± 11−35 ± 25<0.01 Moderate/strong14 ± 180.0 ± 0.0−14 ± 17<0.05 Total57 ± 288.3 ± 11−49 ± 24<0.001H-score71 ± 388 ± 11−63 ± 33<0.001Results are presented as means ± SD.∗ The differences between Rv.C populations are compared by paired Student's t-test. Open table in a new tab Figure 2Discrimination between active or arrested Rv.Ss. Double immunostaining was performed for TRAcP (red) and CD56 (A and B), osterix (C–E), and SMA (F–H) (all brown) on sections of the femur head of PMO patients (A and B), and iliac biopsy specimens from controls (C–H). Masson's trichrome staining was performed on sections of transiliac biopsy specimens of PMO patients (I and J). The staining revealed Rv.Ss (dotted lines) characterized by broken lamellae covered by Rv.Cs (yellow arrows). Their elongated nuclei were visible in Masson trichrome and in osterix and SMA staining. The staining showed that Rv.Cs occupied ES either next to TRAcP+ OCs (A, C, F, and I) (arrowheads) and osteoid (OS) (D, G, and I) or away from OCs and OS (B, E, H, and J). The former two were called active Rv.Ss (Ac.Rv.Ss), and the latter arrested Rv.Ss (Ar.Rv.Ss). The differences in osterix and SMA levels depended on the type of Rv.S: osterix is stronger on active Rv.S next to OS (D) than next to OC (C), and almost absent on arrested Rv.S (E), whereas SMA is absent on active Rv.S next to OS (G), strong next to OCs (F), and weak on arrested Rv.S (H). Fifty sections taken adjacent to those shown in I and J allowed us to see these surfaces in their 3D environment (K and L). The 3D reconstructions of these areas highlight the close proximity of active Rv.S (green) to OS (blue) and OC (red) (K), and, in contrast, the limited OS (blue) and absence of Oc.S in the neighborhood of arrested Rv.S (green) (L). The images inserted into the 3D reconstructions are the respective images from I and J. Scale bars: 50 μm (A–L).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results are pre
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