Voltar
Artigo Revisado por pares
BIBTEX RIS

Bone Matrix, Cellularity, and Structural Changes in a Rat Model with High-Turnover Osteoporosis Induced by Combined Ovariectomy and a Multiple-Deficient Diet

2014; Elsevier BV; Volume: 184; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2013.11.011

ISSN

1525-2191

Autores

Parameswari Govindarajan, Wolfgang Böcker, Thaqif El Khassawna, Marian Kampschulte, Gudrun Schlewitz, Britta Huerter, Ursula Sommer, Lutz Dürselen, Anita Ignatius, Natali Bauer, Gábor Szalay, Sabine Wenisch, Katrin Susanne Lips, Reinhard Schnettler, Alexander C. Langheinrich, Christian Heiß,

Tópico(s)

Bone Metabolism and Diseases

Resumo

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro–computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro–computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants. In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro–computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro–computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants. CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Osteoporosis, a progressive skeletal disorder defined by low bone mass and deteriorated bone microarchitecture, predominantly affects postmenopausal women.1Jagtap V.R. Ganu J.V. Nagane N.S. BMD and serum intact osteocalcin in postmenopausal osteoporosis women.Indian J Clin Biochem. 2011; 26: 70-73Crossref PubMed Scopus (55) Google Scholar, 2Audran M. Jakob F.J. Palacios S. Brandi M.L. Bröll H. Hamdy N.A. McCloskey E.V. A large prospective European cohort study of patients treated with strontium ranelate and followed up over 3 years.Rheumatol Int. 2013; 33: 2231-2239Crossref PubMed Scopus (28) Google Scholar Postmenopausal osteoporosis is characterized by enhanced activation frequency of bone remodeling along with enhanced algebraic difference between bone formation and resorption phases.3Lerner U.H. Bone remodeling in post-menopausal osteoporosis.J Dent Res. 2006; 85: 584-595Crossref PubMed Scopus (246) Google Scholar The skeletal sites of proximal femur, proximal humerus, hip, and vertebral column are more prone to osteoporotic fractures.4Lill C.A. Gerlach U.V. Eckhardt C. Goldhahn J. Schneider E. Bone changes due to glucocorticoid application in an ovariectomized animal model for fracture treatment in osteoporosis.Osteoporos Int. 2002; 13: 407-414Crossref PubMed Scopus (78) Google Scholar Estrogen, vitamin D, and calcium deficiency mainly influence the osteoporotic fractures in elderly women. A large European study of 8532 postmenopausal women with vitamin D inadequacy indicated 97% to be osteoporotic.5Bruyère O. Malaise O. Neuprez A. Collette J. Reginster J.Y. Prevalence of vitamin-D inadequacy in European postmenopausal women.Curr Med Res Opin. 2007; 23: 1939-1944Crossref PubMed Scopus (81) Google Scholar In another study, 82% of osteoporotic patients exhibited inadequate calcium intake with less than the recommended dosage of 1000 mg per day.6Sunyecz J.A. The use of calcium and vitamin-D in the management of osteoporosis.Ther Clin Risk Manag. 2008; 4: 827-836Crossref PubMed Google Scholar Furthermore, phosphorus deficiency is yet another marker of general nutritional inadequacy in elderly patients, causing increased fracture risk.7Nieves J.W. Osteoporosis: the role of micronutrient.Am J Clin Nutr. 2005; 81: 1232-1239Crossref PubMed Scopus (231) Google Scholar Menopause is associated with a sustained increase in calcium excretion.8Nordin B.E. Calcium and osteoporosis.Nutrition. 1997; 13: 664-686Abstract Full Text PDF PubMed Scopus (283) Google Scholar Vitamin D deficiency causes a decrease in calcium absorption. This, in turn, disturbs calcium homeostasis, elevates parathyroid hormone (PTH), and increases bone resorption and bone remodeling rates.9Heaney R.P. Vitamin-D and calcium interactions functional outcomes.Am J Clin Nutr. 2008; 88: 541-544Crossref PubMed Google Scholar, 10Meyer H.E. Falch J.A. Søgaard A.J. Haug E. Vitamin-D deficiency and secondary hyperparathyroidism and the association with bone mineral density in persons with Pakistani and Norwegian background living in Oslo, Norway, The Oslo Health Study.Bone. 2004; 35: 412-417Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Current drugs for osteoporotic therapy are either anti-resorptive or anabolic.11Mosekilde L. Tørring O. Rejnmark L. Emerging anabolic treatments in osteoporosis.Curr Drug Saf. 2011; 6: 62-74Crossref PubMed Scopus (34) Google Scholar, 12Deal C. Potential new drug targets for osteoporosis.Nat Clin Pract Rheumatol. 2009; 5: 20-27Crossref PubMed Scopus (149) Google Scholar, 13Bilezikian J.P. Combination anabolic and antiresorptive therapy for osteoporosis: opening the anabolic window.Curr Osteoporos Rep. 2008; 6: 24-30Crossref PubMed Scopus (91) Google Scholar The efficacy of anti-osteoporotic treatments evidently improved when combined with combinatorial supplementation of calcium and vitamin D, rather than the individual supplementation of either vitamin D or calcium.14Avenell A. Gillespie W.J. Gillespie L.D. O’Connell D. Vitamin-D and vitamin-D analogues for preventing fractures associated with involutional and post-menopausal osteoporosis.Cochrane Database Syst Rev. 2005; 20: CD000227Google Scholar Furthermore, supplementation of calcium as calcium phosphate is preferable in suppressing the phosphorus deficiency.15Sharpiro R. Heaney R.P. Co-dependence of calcium and phosphorus for growth and bone development under conditions of varying deficiency.Bone. 2003; 32: 532-540Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Osteoporosis has study limitations in humans because of its slow progression.16Turner A.S. Animal models of osteoporosis - necessity and limitations.Eur Cell Mater. 2001; 1: 66-81Crossref PubMed Google Scholar Therefore, efficient animal models mimicking human osteoporotic conditions are needed. Studies have performed ovariectomy (OVX) on rats for either osteoporotic induction or fracture healing.17Wang M.L. Massie J. Perry A. Garfin S.R. Kim C.W. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements.Spine J. 2007; 7: 466-474Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 18Tatehara S. Miyamoto Y. Takechi M. Momota Y. Yuasa T. Osteoporosis influences the early period of the healing after distraction osteogenesis in a rat osteoporotic model.J Craniomaxillofac Surg. 2011; 39: 2-9Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 19Kalu D.N. Liu C.C. Hardin R.R. Hollis B.W. The aged rat model of ovarian hormone deficiency bone loss.Endocrinology. 1989; 124: 7-16Crossref PubMed Scopus (346) Google Scholar, 20Yamauchi H. Kushida K. Yamazaki K. Inoue T. Assessment of spine bone mineral density in ovariectomized rats using DXA.J Bone Miner Res. 1995; 10: 1033-1039Crossref PubMed Scopus (51) Google Scholar, 21Wronski T.J. Dann L.M. Scott K.S. Cintrón M. Long-term effects of ovariectomy and aging on the rat skeleton.Calcif Tissue Int. 1989; 45: 360-366Crossref PubMed Scopus (397) Google Scholar However, the classic OVX rat model does not consider the highly prevalent nutritional deficiencies in elderly women.22Melhus G. Solberg L.B. Dimmen S. Madsen J.E. Nordsletten L. Reinholt F.P. Experimental osteoporosis induced by ovariectomy and vitamin-D deficiency does not markedly affect fracture healing in rats.Acta Orthop. 2007; 78: 393-403Crossref PubMed Scopus (47) Google Scholar Few studies have combined OVX with either calcium deficiency23Zhang Y. Dong X.L. Leung P.C. Wong M.S. Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet.Bone. 2009; 44: 46-52Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 24Han S.M. Szarzanowicz T.E. Ziv I. Effect of ovariectomy and calcium deficiency on the ultrasound velocity, mineral density and strength in the rat femur.Clin Biomech. 1998; 13: 480-484Abstract Full Text PDF PubMed Scopus (17) Google Scholar or, rarely, vitamin D deficiency.22Melhus G. Solberg L.B. Dimmen S. Madsen J.E. Nordsletten L. Reinholt F.P. Experimental osteoporosis induced by ovariectomy and vitamin-D deficiency does not markedly affect fracture healing in rats.Acta Orthop. 2007; 78: 393-403Crossref PubMed Scopus (47) Google Scholar OVX combined with a low-calcium diet exhibited decreased bone mineral density (BMD), cortical thinning, and deteriorated trabecular architecture.17Wang M.L. Massie J. Perry A. Garfin S.R. Kim C.W. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements.Spine J. 2007; 7: 466-474Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Furthermore, combined OVX and deficient vitamin D reduced femoral neck strength.25Kaastad T.S. Reikerås O. Halvorsen V. Falch J.A. Obrant K.J. Nordsletten L. Vitamin-D deficiency and ovariectomy reduced the strength of the femoral neck in rats.Calcif Tissue Int. 2001; 69: 102-108Crossref PubMed Scopus (26) Google Scholar An additive effect on bone loss was reported by combined OVX-deficient calcium or OVX-deficient vitamin D.25Kaastad T.S. Reikerås O. Halvorsen V. Falch J.A. Obrant K.J. Nordsletten L. Vitamin-D deficiency and ovariectomy reduced the strength of the femoral neck in rats.Calcif Tissue Int. 2001; 69: 102-108Crossref PubMed Scopus (26) Google Scholar, 26Shen V. Birchman R. Xu R. Lindsay R. Dempster D.W. Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats.Bone. 1995; 16: 149-156Abstract Full Text PDF PubMed Scopus (91) Google Scholar Such combined studies rarely used mature rats (>11 months),23Zhang Y. Dong X.L. Leung P.C. Wong M.S. Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet.Bone. 2009; 44: 46-52Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and have mostly used rats with persistent bone growth (3 to 6 months).22Melhus G. Solberg L.B. Dimmen S. Madsen J.E. Nordsletten L. Reinholt F.P. Experimental osteoporosis induced by ovariectomy and vitamin-D deficiency does not markedly affect fracture healing in rats.Acta Orthop. 2007; 78: 393-403Crossref PubMed Scopus (47) Google Scholar, 25Kaastad T.S. Reikerås O. Halvorsen V. Falch J.A. Obrant K.J. Nordsletten L. Vitamin-D deficiency and ovariectomy reduced the strength of the femoral neck in rats.Calcif Tissue Int. 2001; 69: 102-108Crossref PubMed Scopus (26) Google Scholar, 26Shen V. Birchman R. Xu R. Lindsay R. Dempster D.W. Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats.Bone. 1995; 16: 149-156Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 27Melhus G. Brorson S.H. Baekkevold E.S. Andersson G. Jemtland R. Olstad O.K. Reinholt F.P. Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin-D-depleted rats.Calcif Tissue Int. 2010; 87: 77-89Crossref PubMed Scopus (9) Google Scholar Furthermore, the observational period after OVX with nutrient deficiency rarely exceeded 3 months.22Melhus G. Solberg L.B. Dimmen S. Madsen J.E. Nordsletten L. Reinholt F.P. Experimental osteoporosis induced by ovariectomy and vitamin-D deficiency does not markedly affect fracture healing in rats.Acta Orthop. 2007; 78: 393-403Crossref PubMed Scopus (47) Google Scholar, 23Zhang Y. Dong X.L. Leung P.C. Wong M.S. Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet.Bone. 2009; 44: 46-52Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 25Kaastad T.S. Reikerås O. Halvorsen V. Falch J.A. Obrant K.J. Nordsletten L. Vitamin-D deficiency and ovariectomy reduced the strength of the femoral neck in rats.Calcif Tissue Int. 2001; 69: 102-108Crossref PubMed Scopus (26) Google Scholar, 26Shen V. Birchman R. Xu R. Lindsay R. Dempster D.W. Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats.Bone. 1995; 16: 149-156Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 27Melhus G. Brorson S.H. Baekkevold E.S. Andersson G. Jemtland R. Olstad O.K. Reinholt F.P. Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin-D-depleted rats.Calcif Tissue Int. 2010; 87: 77-89Crossref PubMed Scopus (9) Google Scholar Also, the characterizations were mostly restricted to dual-energy X-ray absorptiometry (DXA), micro–computed tomography (micro-CT), serum parameters, and biomechanics. Long-term observational studies of combined effects exceeding the bone growth phase better address the human conditions.26Shen V. Birchman R. Xu R. Lindsay R. Dempster D.W. Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats.Bone. 1995; 16: 149-156Abstract Full Text PDF PubMed Scopus (91) Google Scholar In addition, biomechanical competence of bone, a matrix of mineral and organic materials, depends on both its material and geometric properties.28Jiang Y. Zhao J. Liao E.Y. Dai R.C. Wu X.P. Genant H.K. Application of micro-CT assessment of 3-D bone microstructure in preclinical and clinical studies.J Bone Miner Metab. 2005; 23: 122-131Crossref PubMed Scopus (119) Google Scholar In osteoporotic bone, the newly synthesized extracellular matrix (ECM) showed an altered or decreased mineral/matrix ratio.29Saito M. Fujii K. Soshi S. Tanaka T. Reductions in degree of mineralization and enzymatic collagen cross-links and increases in glycation-induced pentosidine in the femoral neck cortex in cases of femoral neck fracture.Osteoporos Int. 2006; 17: 986-995Crossref PubMed Scopus (185) Google Scholar, 30Orkoula M.G. Vardaki M.Z. Kontoyannis C.G. Study of bone matrix changes induced by osteoporosis in rat tibia using Raman spectroscopy.Vib Spectrosc. 2012; 63: 404-408Crossref Scopus (24) Google Scholar On the other hand, no animal model exists that addresses the noticed nutrient deficiency and estrogen deficiency seen in postmenopausal elderly women. In addition, there is no continuous long-term information about changes in structure and cellular and bone matrix content in osteoporotic animal models. We present herein, for the first time to our knowledge, the effect of a multiple-deficient diet in combination with estrogen deficiency in a rat model. The diet was mainly deficient in calcium, vitamin D, and phosphorus. This rat model aims to mimic postmenopausal osteoporosis associated with nutrient deficiency in elderly women. Our study is a long-term observation (14 months) after ovariectomy. We analyzed changes in bone formation and resorption at cellular and molecular levels. Furthermore, we also investigate the influence of the treatment on bone mineralization, matrix composition, and their distribution through molecular and histological analysis. Finally, the effects of the combined treatment on bone microarchitecture and biomechanical competence are presented herein. The 2.5-month-old female Sprague-Dawley rats (N = 70) were purchased from Charles River (Sulzfeld, Germany). Animals were acclimatized for 1 month before the experimental procedures. The animals and all of the experimental procedures were approved by German animal protection laws of district government Regierungspräsidium Giessen (89/2009) and Giessen 20/10-Nr.A31/2009. Animals were grouped into three as control, sham, and ovariectomy and diet (OVX-Diet). At 3.5 months of age, the sham group underwent laparotomy while the OVX-Diet group underwent ovariectomy bilaterally after being anesthetized with an i.p. injection of 62.5 mg/kg body weight ketamine (Bela-pharm GmbH & Co KG, Vechta, Germany) and 7.5 mg/kg body weight xylazine (CEVA Tiergesundheit GmbH, Düsseldorf, Germany). Sham rats received normal feed (C1000), whereas OVX-Diet rats were fed with normal feed up to 2 weeks after surgery, after which they were fed with a deficient diet (C1034, deficient mainly in calcium, phosphorus, and vitamin D) (Altromin Spezialfutter GmbH, Lage, Germany). The animals of the control group were euthanized immediately, whereas sham and OVX-Diet groups were analyzed at 3 (M3), 12 (M12), and 14 (M14) months after laparotomy or after OVX-Diet treatment. Bone samples [left humerus, lumbar vertebrae (L1), left femur, right femur, left tibia, and right tibia] devoid of soft tissues were isolated after animal euthanasia. Left humerus and L1 bone samples were stored in RNA later (AM7021; Life Technologies, Darmstadt, Germany) at −80°C for mRNA expression analysis. Right tibia and femur were frozen (−20°C) for biomechanics. Left tibia and femur were used for micro-CT and histochemical analysis, respectively. Femur bone samples were fixed in 4% paraformaldehyde, decalcified (10% EDTA in 0.3 mol/L Tris buffer), and embedded in paraffin. Paraffin sections (5 μm thin) were used for the following staining parameters (TRAP, ALP, IHC). Tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry (N = 6 per group per time point) to evaluate osteoclasts was performed as described previously.31Blumer M.J. Hausott B. Schwarzer C. Hayman A.R. Stempel J. Fritsch H. Role of tartrate-resistant acid phosphatase (TRAP) in long bone development.Mech Dev. 2012; 129: 162-176Crossref PubMed Scopus (48) Google Scholar The following reagents were used: sodium acetate, sodium tartrate (Merck, Darmstadt, Germany), fast red, naphthol-AS-TR-phosphate, N,N-dimethyl formamide (Sigma Aldrich, Steinheim, Germany), and hematoxylin (Fa Shandon, Thermo Scientific, Bonn, Germany). Alkaline phosphatase (ALP) enzyme histochemistry (N = 6 per group per time point) was performed to evaluate osteoblasts containing the following reagents. We used 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate (KPL, Gaithersburg, MD) and nuclear fast red aluminum sulfate solution (Carl Roth + Co KG, Karlsruhe, Germany). Three rats per group per time point were characterized with the following antibodies: mouse anti-osteocalcin (MAB1419; R&D Systems, Wiesbaden-Nordenstadt, Germany), mouse anti-fibronectin (DM300; Acris, Herford, Germany), goat anti-biglycan (ab58562; Abcam, Cambridge, MA), rabbit anti–matrix metallopeptidase (MMP) 9 (ab76003; Abcam), and rabbit anti-tenascin C (ab108930; Abcam). The detection system applied was through an ELITE vectastain ABC kit and novared solution (Vector Laboratories, Inc., Burlingame, CA), with hematoxylin as the counterstain (Fa Shandon, Thermo Scientific). Undecalcified, polymethyl methacrylate (PMMA)–embedded femur bone sections (N = 2 to 3 per OVX-Diet group per time point) were stained with Von Kossa/Van Gieson staining containing silver nitrate, methyl green (Sigma Aldrich), sodium carbonate, 37% formaldehyde, sodium thiosulfate (Merck, Darmstadt, Germany), and Van Gieson (Artikel-Nr. 2E-050; Chroma, Münster, Germany). Metaphyseal bone regions were randomly imaged for each paraffin section with a photographic microscope Axioplan 2 with photomodule Axiophot 2 (Carl Zeiss AG, Jena, Germany) and a DC500 camera (Leica Microsystems Ltd, Heerbrugg, Switzerland) with Leica IM1000 software version 4.0 (Leica Microsystems Imaging Solutions Ltd, Cambridge, UK), for both TRAP and ALP, with at least 15 images at ×40 magnification. Trabecular surface (mm) and osteoclast count (N) were determined for TRAP, whereas trabecular surface (mm) and osteoblast surface (mm) were measured for ALP. All analyses were performed using ImageJ software version 1.45 (NIH, Bethesda, MD). The quantitative RT-PCR (RT-qPCR) was performed to analyze the differential mRNA expression of fibronectin (FN), COL1A1, MMP9, MMP14, biglycan (BGN), carbonic anhydrase II (CA2), tenascin C (TNC), low density lipoprotein receptor-related protein 5 (LRP5), and cathepsin K (CTSK) of control (M0), sham, and OVX-Diet rats (M3, M12, and M14), both in humerus and L1 vertebral body (N = 7 to 10 per group per time point). Total RNA was extracted using TRIzol manufacturer’s protocol (Invitrogen, Carlsbad, CA). Briefly, 1 μg of RNA was reverse transcribed to cDNA with a QuantiTect Reverse Transcriptase Kit (Qiagen, Hilden, Germany). Real-time analysis was done with a Bio-Rad iQ5 PCR cycler (Bio-Rad, Paris, France) by a two-step cycling protocol using a Quantifast SYBR Green RT-PCR kit (Qiagen). B-2 microglobulin was used as the housekeeping gene. Relative mRNA expressions were analyzed for each gene after normalization with the housekeeping gene. The primer sequences are given in Table 1.Table 1Primer Sequences Used in the StudyNameSymbolAccession no.Primer sequencesSize (bp)FibronectinFNX15906.1ForwardReverse5′-CAGCCCCTGATTGGAGTC-3′5′-TGGGTGACACCTGAGTGAAC-3′73Collagen, type I, α 1COL1A1NM_053304.1ForwardReverse5′-TCCTGACGCATGGCCAAGAA-3′5′-CATAGCACGCCATCGCACAC-3′145Matrix metallopeptidase 9MMP9NM_031055.1ForwardReverse5′-TCCCCAGAGCGTTACTCGCT-3′5′-ACCTGGTTCACCCGGTTGTG-3′144Matrix metallopeptidase 14MMP14NM_031056.1ForwardReverse5′-TGGGCCCAAAGCAGCAACTT-3′5′-AGAGTGACTGGGGTGAGCGT-3′106BiglycanBGNNM_017087.1ForwardReverse5′-ACAACAAGCTGTCCCGGGTG-3′5′-AGCCCATGGGGCAGAAATCG-3′117Carbonic anhydrase IICA2NM_019291.1ForwardReverse5′-GCCCCTGCTGGAATGTGTGA-3′5′-TGAGCTGGACGCCAGTTGTC-3′144Tenascin CTNCNM_053861.1ForwardReverse5′-GCATGGGACAATGAGATGCG-3′5′-TCCCGGATGGTGGTAGATGT-3′119Low-density lipoprotein receptor–related protein 5LRP5NM_001106321.2ForwardReverse5′-TCTGGGTGGATGCAGACCTA-3′5′-GTCGATCCAGTAGAGGTGCC-3′134Cathepsin KCTSKNM_031560.2ForwardReverse5′-GCAGCAGAATGGAGGCATTG-3′5′-TTCAGGGCTTTCTCGTTCCC-3′141β-2 MicroglobulinB2MNM_012512.1ForwardReverse5′-TGTCTCAGTTCCACCCACCT-3′5′-GGGCTCCTTCAGAGTGACG-3′191 Open table in a new tab Microarchitecture of left tibias of M0, sham, and OVX-Diet (M3, M12, and M14) rats was evaluated by scanning in micro-CT (SkyScan1072_80 kV; Kontich, Antwerp, Belgium), as described previously,32Govindarajan P. Khassawna T. Kampschulte M. Böcker W. Huerter B. Dürselen L. Faulenbach M. Heiss C. Implications of combined ovariectomy and glucocorticoid (dexamethasone) treatment on mineral, microarchitectural, biomechanical and matrix properties of rat bone.Int J Exp Pathol. 2013; 94: 387-398Crossref PubMed Scopus (34) Google Scholar with a maximum spatial resolution of 8 μm at 10% modulation transfer function. Samples were positioned on a computer-controlled rotation stage and scanned 180° around the vertical axis in rotation steps of 0.45° at 75 kV. Tibia was scanned en bloc with an isotropic spatial resolution of (9 μm)3 voxel size. Raw data were reconstructed with a modified Feldkamp cone-beam reconstruction modus, resulting in two-dimensional cross-sectional images with an 8-bit gray-scale resolution. The following parameters were measured: relative bone/trabecular volume (BV/TV%), trabecular separation (Tb.Sp), trabecular number (Tb.N), and structure model index (SMI). Three-point bending test of right femurs (N = 5 to 7 per group per time point) was determined, as described previously,33Röntgen V. Blakytny R. Matthys R. Landauer M. Wehner T. Göckelmann M. Jermendy P. Amling M. Schinke T. Claes L. Ignatius A. Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator.J Orthop Res. 2010; 28: 1456-1462Crossref PubMed Scopus (94) Google Scholar in a standard materials testing machine (Z010; Zwick, Ulm, Germany). The distal end of femurs was embedded in PMMA, and they were placed in a customized three-point bending rig at a support width of 32 mm. A central bending load was applied at a displacement rate of 1 mm/minute. After two preload cycles up to 5 N, a third cycle up to 20 N was performed. The bending stiffness (N/mm) was determined from the third cycle. Finally, a load to failure test revealed the breaking load (N) of the femora. Torsional stiffness of right tibias was also determined as described previously.29Saito M. Fujii K. Soshi S. Tanaka T. Reductions in degree of mineralization and enzymatic collagen cross-links and increases in glycation-induced pentosidine in the femoral neck cortex in cases of femoral neck fracture.Osteoporos Int. 2006; 17: 986-995Crossref PubMed Scopus (185) Google Scholar Briefly, both ends of tibia were embedded in PMMA and connected to the testing machine (Z10; Zwick). A vertical displacement was applied to a lever arm (L = 50 mm) at a quasi-static displacement rate of 20 mm/minute. The required force to displace the lever arm was registered by a 50 N load cell (KAP-Z; A.S.T., Dresden Germany). After a preload cycle, the test was repeated three times up to a maximum vertical force of 1 N and the torsional stiffness (Nmm/°) was calculated from the last loading cycle between 5 and 40 Nmm torsional moment. Finally, the bones were tested to failure, and the breaking moment was determined. Statistical analysis (Graphpad Prism version 5; La Jolla, CA) for enzyme histochemistry, RT-qPCR, and biomechanics was done using the Mann-Whitney test to determine significance between groups and the Kruskal-Wallis test for differences across time points in a single group. Furthermore, micro-CT statistics involved two-way analysis of variance accompanied by Bonferroni’s multiple-comparison test. Our data up to 3 months [TRAP, ALP (N = 3, preliminary results), micro-CT, and biomechanics] are toward publication (in press),34El Khassawna T. Böcker W. Govindarajan P. Schliefke N. Hürter B. Kampschulte M. Schlewitz G. Alt V. Lips K.S. Faulenb

Referência(s)