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. 2013 May;54(1):83–91. doi: 10.1016/j.bone.2013.01.036

Alteration of proteoglycan sulfation affects bone growth and remodeling

Benedetta Gualeni a,1, Marie-Christine de Vernejoul b,c, Caroline Marty-Morieux b,c, Fabio De Leonardis a, Marco Franchi d, Luca Monti a, Antonella Forlino a, Pascal Houillier e, Antonio Rossi a,⁎, Valerie Geoffroy b,c
PMCID: PMC3607217  PMID: 23369989

Abstract

Diastrophic dysplasia (DTD) is a chondrodysplasia caused by mutations in the SLC26A2 gene, leading to reduced intracellular sulfate pool in chondrocytes, osteoblasts and fibroblasts. Hence, proteoglycans are undersulfated in the cartilage and bone of DTD patients. To characterize the bone phenotype of this skeletal dysplasia we used the Slc26a2 knock-in mouse (dtd mouse), that was previously validated as an animal model of DTD in humans.

X-rays, bone densitometry, static and dynamic histomorphometry, and in vitro studies revealed a primary bone defect in the dtd mouse model.

We showed in vivo that this primary bone defect in dtd mice is due to decreased bone accrual associated with a decreased trabecular and periosteal appositional rate at the cell level in one month-old mice. Although the osteoclast number evaluated by histomorphometry was not different in dtd compared to wild-type mice, urine analysis of deoxypyridinoline cross-links and serum levels of type I collagen C-terminal telopeptides showed a higher resorption rate in dtd mice compared to wild-type littermates. Electron microscopy studies showed that collagen fibrils in bone were thinner and less organized in dtd compared to wild-type mice. These data suggest that the low bone mass observed in mutant mice could possibly be linked to the different bone matrix compositions/organizations in dtd mice triggering changes in osteoblast and osteoclast activities.

Overall, these results suggest that proteoglycan undersulfation not only affects the properties of hyaline cartilage, but can also lead to unbalanced bone modeling and remodeling activities, demonstrating the importance of proteoglycan sulfation in bone homeostasis.

Abbreviations: BER, bone elongation rate; BFR, bone formation rate; BMC, bone mineral content; BMD, bone mineral density; BrdU, 5-bromo-2′-deoxyuridine; CTX, C-terminal telopeptides of type I collagen; DEXA, dual energy X-ray absorptiometry; DLS/BS, double labeled surface per bone surface; DPD, deoxypyridinoline; DTD, diastrophic dysplasia; DTDST, diastrophic dysplasia sulfate transporter; FCS, fetal calf serum; MAR, mineral apposition rate; M-CSF, macrophage colony-stimulating factor; P, postnatal day; PBS, phosphate buffer saline; PTH, parathyroid hormone; RANK-L, receptor activator of nuclear factor kappa-B ligand; SLC26A2, solute carrier family 26 member 2; TRAP, tartrate resistant acid phosphatase

Keywords: Diastrophic dysplasia, Proteoglycan, Bone histomorphometry, Animal models, Osteoclasts, Osteoblasts

Highlights

► The osteopenic phenotype in a mouse model of proteoglycan undersulfation has been characterized. ► In vivo and in vitro studies revealed a primary bone defect in the dtd mouse model. ► Low bone mass in mutant mice is linked to bone matrix alterations triggering changes in osteoblast and osteoclast activities. ► Electron microscopy showed that collagen fibrils were thinner and less organized in mutant compared to wild-type mice. ► Results demonstrate that the SLC26A2 gene not only affects chondrogenesis, but also leads to unbalanced bone modeling and remodeling activities.

Introduction

The diastrophic dysplasia sulfate transporter (DTDST, also known as SLC26A2) is a sulfate/chloride antiporter, widely expressed on the plasma membrane of many cell types, including fibroblasts, chondrocytes and osteoblasts [1].

Functional defects of the SLC26A2 can cause a reduction in the intracellular sulfate pool, leading to synthesis and secretion of undersulfated proteoglycans [2]. Proteoglycan undersulfation can result in altered architecture and mechanical properties of the extracellular matrix [3]. The consequences of these alterations are most evident at the cartilage level, since cartilage is a tissue very rich in proteoglycans that in normal conditions are massively sulfated. Thus, defects in the SLC26A2 can cause a chondrodysplastic phenotype. Mutations in the gene encoding for the SLC26A2 are indeed associated with a family of recessively inherited chondrodysplasias that include, in order of increasing severity, a recessive form of multiple epiphyseal dysplasia, diastrophic dysplasia (DTD), atelosteogenesis type 2, and achondrogenesis type 1B [4]. The different clinical phenotypes are related to the residual activity of the sulfate transporter and thus to the resulting degree of proteoglycan undersulfation [2].

We have previously generated a mouse model (dtd mouse) in which the murine homologue of the SLC26A2 gene was knocked-in with a mutation previously identified in a DTD patient. Homozygous mutant mice were shown to reproduce some of the clinical, morphological and biochemical features of DTD in humans, being characterized by growth retardation, skeletal dysplasia, joint contractures and reduced viability. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, proteoglycan undersulfation in articular cartilage and delayed secondary ossification center formation. Impaired sulfate uptake was observed in chondrocytes, osteoblasts and fibroblasts demonstrating the generalized nature of the sulfate uptake defect [5]. Consistent with the uptake defect, proteoglycan undersulfation was observed also in the growth plate of homozygous mutant mice, causing altered histomorphometric parameters, reduced chondrocyte proliferation, and altered Ihh signaling pathway [6]. However bone studies demonstrated that skeletal defects were not restricted to the articular cartilage or to the growth plate. The sulfate uptake defect was detected also in osteoblasts, and chondroitin sulfate proteoglycans from the femoral diaphysis of mutant mice were slightly but significantly undersulfated between postnatal days P7 and P60. Moreover, signs of early osteoporosis of long bones were detected in dtd mice at P60 [5]. These data demonstrated that a bone phenotype which has never been investigated in DTD patients was present in the dtd mouse.

When studying chondrodysplasias, attention is particularly focussed on the articular and growth plate cartilage, since common features of chondrodysplasias in humans and mice include retarded skeletal development, failure of growth plate chondrocytes to undergo the normal proliferation and maturation pathway, and osteoarthritis [7–15]. Usually patients do not appear to have bone problems such as bone fragility or osteoporosis and for these reasons bone studies are scarce. Nevertheless, the bone phenotype has been analyzed extensively in a few chondrodysplasia mouse models [16–21].

In order to better characterize the bone phenotype in dtd mice and to investigate whether it is a consequence of the cartilage defect or a primary bone defect, we performed radiographies, dual-energy X-ray absorptiometry, and static and dynamic histomorphometry on the long bones of dtd mice, as well as in vitro studies on cultured osteoblasts and osteoclasts, and analysis of markers of altered bone homeostasis in serum and urine.

Our results suggest that the bone phenotype in dtd mice is a primary bone defect, which is not due to defects in osteoblast mineralization, osteoclast differentiation or systemic alterations, but to increased degradation of the altered organic bone matrix. Ultimately, these results show an important role for proteoglycan sulfation in bone homeostasis.

Material and methods

Animals

The dtd mouse is a “knock-in” for a c1184t transition causing an A386V substitution in the eighth transmembrane domain of the SLC26A2, which strongly reduces the activity of the transporter. Homozygous mutant mice (dtd) show a chondrodysplastic phenotype that recapitulates essential aspects of human DTD [5]. In this study, 1 and 2 month-old male wild-type and dtd mice with a mixed C57Bl/6J × 129/SV background were used. Older animals couldn't be analyzed due to the reduced life span of dtd mice [5].

Genomic DNA was isolated from mouse tail clips and genotyping to distinguish homozygous mutant animals from heterozygous and wild-type littermates was then performed either by PCR or by Southern blotting.

Animals were bred with free access to water and standard pelleted food. Care and use of mice for this study were in compliance with relevant animal welfare guidelines approved by the Animal Care and Use Committee of the University of Pavia.

Only male animals were considered in this study, since age-related bone changes are more dramatic in C57BL/6J wild-type females than in male littermates [22].

Radiographies and dual-energy X-ray absorptiometry (DEXA)

Radiographies were performed on the tibiae of 1 (n = 12) and 2 (n = 6) month-old wild-type and dtd males mice using an X-ray cabinet (Faxitron).

DEXA was performed on the tibiae of 1 (n = 12) and 2 (n = 6) month-old wild-type and mutant animals using a PIXImus Mouse Densitometer (Lunar GE Medical Systems) in order to evaluate bone area, bone mineral density (BMD) and bone mineral content (BMC).

Bone histology and histomorphometry

Bone histomorphometry was performed as described elsewhere [23]. Briefly, mice were given two fluorochrome labels by intraperitoneal injection: 1 month-old mice (n = 12) were injected with 20 mg/kg tetracycline (Sigma) 3 days before sacrifice, and with 10 mg/kg calcein (Sigma) the day before sacrifice, while 2 month-old animals (n = 6) were injected with 20 mg/kg tetracycline 4 days before sacrifice, and with 10 mg/kg calcein the day before sacrifice.

Mice were sacrificed 24 h after the last injection. Femora were excised immediately after sacrifice and cleaned from the surrounding soft tissues. Bones were trimmed, and their distal halves were post-fixed in 70% ethanol, dehydrated in graded alcohols at 4 °C, defatted in xylene, and embedded without demineralization in methyl methacrylate.

Coronal sections (5 or 12 μm thick) of the central region of the distal femur were cut parallel to the long axis of the bone, using a SM2500S microtome (Leica) with a tungsten carbide knife. Consecutive sections were used for different purposes. Some of them (5 μm thick) were stained for tartrate resistant acid phosphatase (TRAP) detection using Naphthol AS-TR Phosphate (Sigma) as substrate, and counterstained with 0.5% toluidine blue (pH 4.3). Other 5 μm thick sections were stained with 1% toluidine blue (pH 4.3) for evaluation of bone formation parameters or with the Modified Masson's Trichrome staining for the evaluation of static parameters. Other sections (12 μm thick) were left unstained and analyzed under UV light for evaluation of the dynamic parameters. A mean of 6 sections per animal per staining was analyzed.

All measurements were performed in the secondary spongiosa, in an area of about 1.4 mm2 located about 600 μm away from the growth plate (red squares in Fig. 2, panel A). The histomorphometric parameters were recorded in this standardized area in compliance with the recommendation of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee [24].

Fig. 2.

Fig. 2

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Altered bone architecture in 1 and 2 month-old mutant mice. Modified Masson's Trichrome staining (panel A) of the distal femur of 1 and 2 month-old wild-type (wt) and mutant (dtd) animals shows that the trabecular bone architecture is markedly altered in the secondary spongiosa of dtd mice. All bone measurements have been performed in a standardized area of the secondary spongiosa, delimitated by red rectangles (1.4 mm × 1 mm). Trabecular bone volume (panel B), trabecular separation (panel C), trabecular thickness (panel D) and cortical thickness (panel E) in wild-type (wt, patterned columns) and mutant (dtd, white columns) mice are presented, together with bone diameter (panel F) and marrow diameter (panel G), showing alterations in the bone architecture of mutant mice at the two age points considered. Columns represent mean ± SEM (a, p < 0.0001; b, p < 0.002; c, p < 0.05).

Trabecular bone volume, trabecular thickness, trabecular number, trabecular separation, bone diameter, marrow diameter, cortical thickness, and osteoid thickness were measured using a semiautomatic image-analysis system (MicroVision) linked to a light microscope. The osteoclast, osteoblast and osteoid surfaces were measured using an objective eyepiece Leitz integrateplatte II. The dynamic parameters were measured in 12 μm thick unstained sections that were examined under UV light. The mineral apposition rate and the bone elongation rate were measured using the semiautomatic image-analysis system linked to a UV light microscope. The mineralizing surfaces were measured in the same area using the objective eyepiece Leitz integrateplatte II.

Scanning electron microscopy

Tibiae of 1 and 2 month-old wild-type and dtd mice (n = 4) were excised immediately after sacrifice, cleaned from the surrounding soft tissues and fixed for 24 h in 4% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. Tibiae were then isolated and fractured by a knife following a sagittal plane in the central region. After washing in 0.1 M sodium cacodylate buffer, pH 7.4, the samples were first incubated in 5% sodium hypochlorite to remove bone marrow for 10 min at room temperature, and then treated with a 35% phosphoric acid gel (Temrex Gel Etch, Temrex Corporation, New York, USA) for 60 s to expose the collagen fibrils on the bone surface. Sectioned tibiae were dehydrated in a graded series of alcohol and then in hexamethyldisilazane. Finally, they were mounted on metal stubs, coated with a layer of gold in an Emitech K550 sputter-coater to be observed in a scanning electron microscope (Philips 515, Eindhoven, The Netherlands) operating in secondary-electron mode.

Serum and urine biochemistry

Blood samples were collected from 1 and 2 month-old wild-type and mutant animals in the morning, after overnight fasting from tail vein. Serum was obtained from centrifugation of whole blood samples at 10,000 ×g for 10 min at 4 °C.

Routine techniques were used at the Hôpital Européen Georges Pompidou (Paris, France) to determine Ca2 + and parathyroid hormone (PTH) concentrations in serum of 1 month-old mice (n = 5).

To assess bone resorption in vivo, the deoxypyridinoline (DPD) cross-links in the urine of 1 and 2 month-old wild-type and dtd mice were evaluated using the MicroVue™ EIA kit (Quidel Corp.) according to the manufacturer's suggestions; values were normalized to creatinine concentration using a standard colorimetric Jaffe method. All samples (n = 10) were run in double. Furthermore the amount of C-terminal telopeptide of type I collagen (CTX) released in serum of 2 month-old wild-type and mutant mice was evaluated using the RatLaps™ ELISA kit (Nordic Bioscience Diagnostics), according to the manufacturer's suggestions. All samples (n = 20) were run in double.

Osteoblast cultures

To isolate primary osteoblasts, calvaria from 2 or 3 day-old wild-type and dtd mice were aseptically dissected. Parietal bones were isolated and cleaned from the adherent connective tissue. Bones were washed three times in phosphate buffer saline (PBS) (Sigma) containing 1% antibiotic solution (Penicillin/Streptomycin 100 ×, EuroClone) in a shaking bath at 37 °C for 10 min, and then pre-digested 2 times with 200 U/ml collagenase (type IV from Clostridium histolyticum, Sigma) in PBS in a shaking bath at 37 °C for 15 min. Three consecutive digestions were performed incubating bones with 200 U/ml collagenase in PBS at 37 °C for 20 min in a shaking bath. After every digestion, the solution was aspirated carefully and filtered through 70 μm diameter cell strainer (BD Falcon) to recover cells excluding any particles of bone, and centrifuged at 300 ×g for 5 min. Pelleted cells were resuspended in α-MEM (EuroClone) containing 10% heat-inactivated fetal calf serum (HI-FCS, EuroClone) and 1% antibiotics, counted and plated for proliferation and mineralization assays.

Osteoblast proliferation

For osteoblast proliferation assay, primary osteoblasts extracted as described above from wild-type and dtd mice were plated on 4-well Lab-Tek Chamber Slides (4 × 105 cells/well), and incubated for 4 days in α-MEM supplemented with 25 μg/ml ascorbic acid, 10% FCS and 1% antibiotics at 37 °C in 5% CO2, changing medium on day two. Cells were then labeled in the same medium supplemented with 5 μM 5-bromo-2′-deoxyuridine (BrdU) for 4 h. At the end of the labeling period, detection of labeled cells was performed using a specific anti-BrdU antibody (GE Healthcare) and the HistoMouse™ MAX kit (Zymed Laboratories), according to the manufacturer's suggestions. Percentage of cells incorporating BrdU was determined by counting positive nuclei on a total of 3000 random nuclei per genotype in three independent experiments.

Osteoblast phenotype analysis

Primary calvarial osteoblasts extracted as described above were plated on 6 cm diameter Petri dishes at a density of 2 × 105 cells/dish in α-MEM containing 10% HI-FCS and 1% antibiotics and maintained at 37 °C in 5% CO2 until they reached sub-confluence, changing medium every second day. The osteoblastic phenotype of cells was then confirmed using the Alkaline Phosphatase Detection kit (Sigma).

For osteoblast mineralization assay, primary osteoblasts were seeded into T25 flasks (BD Falcon) at a density of 3 × 105 cells/flask in α-MEM containing 10% HI-FCS and 1% antibiotics and maintained at 37 °C in 5% CO2 until they reached confluence (typically after 4 to 6 days), changing medium every second day. Adherent cells were trypsinized and re-plated in 6-well plates (BD Falcon) at a density of 2 × 105 cells/well in the same medium, supplemented with 100 μg/ml ascorbic acid and 5 mM β-glycerophosphate to induce matrix mineralization, and maintained at 37 °C in 5% CO2. The medium was changed every second day and mineralized nodule formation was observed after different culture times (1, 2, 4 and 6 weeks) by Von Kossa staining, according to standard procedures [25].

Osteoclast cultures

Two types of cells were used as a source of osteoclast precursor: spleen cells, obtained from the spleen of 1 month-old wild-type and dtd mice, and bone marrow cells flushed from the femora of 1 month-old wild-type and dtd mice, treated with red blood cells lysis solution (Sigma) as described elsewhere[26]. Both spleen and bone marrow derived cells were resuspended in α-MEM with 10% FCS and 1% antibiotics.

Spleen cells were cultured overnight in 10 cm2 bacterial Petri dishes at 37 °C in 5% CO2. Adherent cells were then trypsinized and plated at a density of 2 × 106 cells/well in 24-well plates in the same medium, in the presence or absence of 25 ng/ml recombinant mouse M-CSF (R&D Systems) and 100 ng/ml recombinant mouse RANK-L (R&D Systems). Cells were incubated for 7 days at 37 °C in 5% CO2 and the medium was changed every second day.

Bone marrow cells were cultured overnight in 6-well plates (1 × 107 cells/well) in the same medium described above in the presence of 5 ng/ml recombinant mouse M-CSF at 37 °C in 5% CO2. Non-adherent cells were then collected and plated at a density of 2 × 106 cells/well in 24-well plates as described for spleen-derived cells. Cells were incubated for 7 days at 37 °C in 5% CO2 and media were changed every second day.

To identify multinucleated osteoclasts, cells were fixed with 10% formaldehyde in PBS (Sigma) and stained for TRAP using the Leukocyte Acid Phosphatase kit (Sigma) according to the manufacturer's instructions.

Statistical analysis

For ex vivo experiments, statistical differences between the means in the different groups tested were evaluated by one-way ANOVA, followed by Fisher's LSD test. For in vitro experiments, statistical differences between the different groups were evaluated using Student's t-test. Results are expressed as means ± SEM. A value of p < 0.05 was considered statistically significant.

Results

Shortening of long bones and reduced bone mass in dtd mice

Radiographic analysis of tibiae from 1 and 2 month-old animals (Fig. 1, panel A) showed that in mutant mice tibiae are more bowed and significantly shorter (27% and 24%, at 1 and 2 months of age respectively) compared to those of wild-type littermates (Fig. 1, panel B). Reduced long bone growth was confirmed by the longitudinal bone elongation rate evaluated on undecalcified sections of the distal femur of 1 and 2 month-old wild-type and dtd mice, labeled in vivo with tetracycline and calcein [27]. The bone elongation rate (mean distance between the proximal traces of the two labels divided by the marker interval) in the metaphyseal cancellous bone is 50% and 45% lower in mutant mice compared to wild-type littermates at 1 and 2 months respectively (Fig. 1, panel C).

Fig. 1.

Fig. 1

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Long bones of mutant mice are shorter and have a lower bone mineral mass compared to control animals. X-ray analysis (panel A) shows that the tibiae of mutant mice (dtd) are shorter and more bowed than normal (wt) at the two age points considered. The tibial length (panel B) is significantly reduced in mutant mice (dtd, white columns) when compared to normal (wt, patterned columns) littermates at the two age points considered, as a result of the significantly decreased bone elongation rate (panel C). Both bone mineral content (panel D) and bone mineral density (panel E) are significantly lower in dtd animals at the two age points considered. Columns represent mean ± SEM (a, p < 0.0001; b, p < 0.002; c, p < 0.05).

Bone densitometry analysis performed on the tibiae of 1 and 2 month-old wild-type and dtd mice showed that not only bone size, but also bone quality is affected in mutant mice. In fact, both bone mineral content (BMC) and bone mineral density (BMD) are significantly lower in mutant animals when compared to wild-type littermates (Figs. 1, panels D and E). Although absolute values of bone length and BMD were lower in dtd compared to wild-type mice, an overall increase in bone mass and size occurred between 1 and 2 months of age in both genotypes.

Altered bone architecture in dtd mice

To determine structural parameters of trabecular bone, we performed static histomorphometry on sections of the undecalcified distal femur of 1 and 2 month-old wild-type and dtd mice, stained with Modified Masson's Trichrome (Fig. 2, panel A). In the secondary spongiosa of dtd mouse long bones, the trabecular bone volume is respectively 69% and 31% lower at 1 and 2 months of age when compared to wild-type littermates (Fig. 2, panel B), and the trabecular separation is significantly higher at the two age points considered compared to wild-type littermates (Fig. 2, panel C). Both trabecular and cortical thickness are significantly lower only at 1 month of age in mutant mice, reaching normal values at 2 months of age (Figs. 2, panels D and E). The bone diameter is respectively 20% and 13% lower in mutant mice at 1 and 2 months of age compared to control animals (Fig. 2, panel F), while the marrow diameter is significantly lower (− 15%) in 1 month-old mutant mice and reach normal values at 2 months of age (Fig. 2, panel G). These results also indicate that gain of bone mass occurs between 1 and 2 months of age at the cortical compartment in both dtd and wild-type mice. At the secondary spongiosa, trabecular thickness was increased between 1 and 2 months of age only in dtd mice.

Bone collagen fibrils in dtd mice are less organized forming an irregular array

In agreement with the histological observations, the bone trabeculae of the secondary spongiosa from tibiae of 1 month-old dtd mice appeared slightly thinner and considerably fewer compared to those from age matched wild-type animals when observed under a scanning electron microscope (data not shown). At high magnifications bone trabeculae of 1 month-old animals showed regions of exposed collagen fibrils. The fibrils of dtd mice showed, on average, a smaller diameter when compared to fibrils of wild-type mice, and in some areas they appeared less densely packed with a more irregular pattern. Tibiae of dtd and wild-type animals at 2 months showed bone trabeculae of similar size with osteoblasts, but the fibrils exposed from the bone trabeculae of dtd mice showed fibrils with a lower diameter and a less organized arrangement when compared to fibrils of wild-type mice (Fig. 3).

Fig. 3.

Fig. 3

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Scanning electron micrographs of the inner bone surface. The collagen fibrils exposed from the bone trabeculae of dtd mice at 1 and 2 months of age are less organized when compared to fibrils of age matched wild-type mice and show a smaller diameter.

Individual osteoblastic activity is mildly decreased in growing dtd mice and restored in young adult dtd mice

Bone formation parameters were evaluated on sections of the undecalcified distal femur of 1 and 2 month-old wild-type and dtd mice, stained with toluidine blue. Osteoid thickness is significantly lower (− 15%) in 1 month-old mutant mice compared to wild-type littermates, but reaches normal values with aging. The osteoid and osteoblast surfaces are normal in dtd mice at both the age points considered (Table 1).

Table 1.

Bone formation parameters in the trabecular bone of 1 and 2 month-old wild-type and mutant mice.

Bone formation 1 month
 2 months
Wild-type dtd Wild-type dtd
Osteoid surface/bone surface (OS/BS; %) 26.3 ± 2.1 30.5 ± 2.6 17.4 ± 2.6 12.1 ± 1.6
Osteoblast surface/bone surface (Ob.S/BS; %) 13.8 ± 1.2 13.7 ± 2.0 5.0 ± 0.7 2.5 ± 1.3
Osteoid thickness (μm) 2.9 ± 0.1 2.5 ± 0.1a 2.3 ± 0.1 2.2 ± 0.1
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Values are shown as mean ± SEM. n = 12 (1 month) or 6 (2 months).

a

Significantly different from wild-types (p < 0.05).

The dynamics of bone formation was assessed on sections of the undecalcified femur of 1 and 2 month-old wild-type and mutant animals, previously labeled in vivo with two different fluorochromes (tetracycline and calcein); dynamic histomorphometry parameters were determined in both trabecular and cortical bones. Concerning trabecular bone (Fig. 4, panel A), we observed that the double labeled surface per bone surface (DLS/BS) in dtd mice is identical to wild-type mice at the two age points considered (Fig. 4, panel B). The mineral apposition rate (MAR) is significantly lower (− 14%) in the trabecular bone of 1 month-old dtd mice compared to wild-type animals, and reaches normal values at 2 months of age (Fig. 4, panel C). The trabecular bone formation rate (BFR) in mutant mice is not significantly different from wild-types at the two age points considered (Fig. 4, panel D). The mineralizing lag time is normal in dtd mice at the two age points considered (data not shown). These data show a reduced individual osteoblast apposition rate in the trabecular bone of 1 month-old dtd mice.

Fig. 4.

Fig. 4

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Dynamic histomorphometric parameters of trabecular bone formation in 1 and 2 month-old wild-type and mutant animals. Double labeled trabeculae from the secondary spongiosa of the femur of 1 and 2 month-old wild-type (wt) and mutant (dtd) mice are shown in panel A (white bar = 10 μm). Double labeled surface per bone surface (panel B), mineral apposition rate (panel C) and the bone formation rate (panel D) were evaluated in wild-type (wt, patterned columns) and mutant (dtd, white columns) mice. These data suggest no osteoblastic or mineralization defects in the trabecular bone of dtd mice. Columns represent mean ± SEM (a, p < 0.0001).

In the periosteum, the bone formation rate at tissue level in 1 month-old dtd mice is identical to controls (BFR) but mineralization appositional rate (MAR) was decreased. At two months of age, periosteal DLS/BS, MAR and BFR are significantly higher (+ 59%, + 19% and + 91% respectively) in dtd mice compared to wild-types (Figs. 5, panel A–C). In the endosteum, the DLS/BS and the BFR are unchanged in mutant animals compared to wild-types at the two age points considered (Figs. 5, panels D and F). The endosteal MAR is normal in 1 month-old mutant mice, but it is higher (+ 30%) in 2 month-old mutant animals compared to age matched controls (Fig. 5, panel E). These results indicate that the increase in cortical thickness between 1 and 2 months of age in dtd mice is due to a sustained bone formation rate at both the endosteum and the periosteum compared to wild-type mice.

Fig. 5.

Fig. 5

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Dynamic histomorphometric parameters of cortical bone formation in 1 and 2 month-old wild-type and mutant animals. The cortical bone from the femur of 1 and 2 month-old wild-type (wt, patterned columns) and mutant (dtd, white columns) mice was analyzed. The double labeled surface per bone surface (panels A and D), mineral apposition rate (panels B and E) and bone formation rate (panels C and F) were evaluated both at the periosteum and at the endosteum compartments respectively. These data suggest no osteoblastic or mineralization defects in the cortical bone of dtd mice. Columns represent mean ± SEM (c, p < 0.05).

To further explore bone formation in dtd mice, osteoblastic activity and mineralization were assessed in vitro on primary osteoblast cultures. Osteoblasts extracted from the calvarial bone of mutant mice proliferate normally (dtd 12.7 ± 5.3%; wild-type 15.2 ± 5.5%; p = 0.2), show the same alkaline phosphatase activity as the cells extracted from control animals, and produce mineralized nodules normally (data not shown).

Seric levels of Ca2 + and parathyroid hormone (PTH) are the same in dtd and wild-type mice

To investigate whether the bone defect observed in dtd mice was related to systemic alterations in mineral homeostasis and to exclude secondary hyperparathyroidism, we measured the Ca2 + and PTH concentrations in sera of 1 month-old wild-type and dtd mice. We observed that there are no significant differences between mutant and control animals at 1 month of age in serum levels of both Ca2 + (1.79 ± 0.30 mM and 1.79 ± 0.26 mM respectively; p = 0.97) and PTH (13.41 ± 2.69 ng/l and 14.52 ± 5.40 ng/l respectively; p = 0.50).

Osteoclast differentiation is similar in dtd and wild-type animals

The osteoclast surface and number were evaluated on sections of the undecalcified distal femur of 1 and 2 month-old wild-type and dtd mice, stained for TRAP detection and counterstained with toluidine blue. We observed that osteoclast surface and osteoclast number are the same in the secondary spongiosa of wild-type and mutant animals (Table 2).

Table 2.

Bone resorption parameters in the trabecular bone of 1 and 2 month-old wild-type and mutant mice.

Bone resorption 1 month
 2 months
Wild-type dtd Wild-type dtd
Osteoclast surface/bone surface (Oc.S/BS; %) 7.5 ± 1.3 5.9 ± 1.9 13.0 ± 5.9 8.1 ± 5.3
Osteoclast number/bone surface (Oc.N/BS; #/mm) 1.01 ± 0.18 1.30 ± 0.36 2.07 ± 0.21 1.85 ± 0.14
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Values are shown as mean ± SEM. n = 12 (1 month) or 6 (2 months).

To determine whether alterations in the DTDST function have a direct effect on osteoclast differentiation, ex vivo osteoclastogenesis was induced and analyzed using osteoclastic progenitors from spleen and bone marrow. After culture in the presence of mouse recombinant M-CSF and mouse recombinant RANK-L for 1 week on plastic dishes, TRAP staining showed that the number of TRAP positive multinucleated cells is not different between genotypes, and indicated no differentiation defect ex vivo (Fig. 6).

Fig. 6.

Fig. 6

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In vitro osteoclast differentiation. Mutant (dtd) and wild-type (wt) mouse osteoclast progenitors originating from spleen cells and bone marrow differentiate equally into osteoclasts when appropriately stimulated. Cells were stained for TRAP activity and counterstained with hematoxylin (panel A, magnification is 50 ×). The number of multinucleated cells per field after 7 days of culture was similar in wild-type (wt, patterned columns) and mutant (dtd, white columns), independently of the source of osteoclast progenitor cells (panel B, n = 7).

Urinary and seric bone parameters indicate increased bone resorption in dtd mice

To study bone turn-over in vivo urine excretion of DPD cross links as an indicator of bone resorption was evaluated in 1 and 2 month-old mice and normalized to creatinine concentration. DPD values were significantly higher in dtd animals compared to wild-type littermates at both age points indicating increased bone resorption in mutant mice (Figs. 7, panels A and B). The data were confirmed by serum levels of CTX, an additional marker of collagen degradation, which were significantly higher (+ 111%) in 2 month-old mutant mice when compared to wild-types (Fig. 7, panel C). Overall, seric and urine bone parameters suggest that bone matrix degradation is higher in dtd mice than in wild-type animals.

Fig. 7.

Fig. 7

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In vivo bone turn-over. Urine DPD cross links and serum CTX levels were measured in mutant (dtd, white columns) and wild-type (wt, patterned columns) mice to study bone resorption. DPD values normalized to the creatinine concentration were significantly higher in dtd animals compared to wt littermates at 1 and 2 months of age (panels A and B) suggesting increased bone resorption. CTX levels, measured at 2 months of age, were significantly higher in the serum of mutant mice (dtd) when compared to wild-type littermates (wt) at 2 months of age, confirming increased bone turn-over in mutant animals (panel C). Columns represent mean ± SEM (a, p < 0.0001; b, p < 0.002).

Discussion

During the characterization of the mouse model of DTD, we had evidence that long bones of dtd mice were osteopenic at 2 months of age. We observed less bone in the secondary spongiosa of mutant mice, with trabeculae that were thinner and poorly connected to each other compared to wild-types [5]. In addition to these histological observations, we showed in the same paper that sulfate uptake in murine dtd osteoblasts was impaired and consequently proteoglycans from mature bone were slightly but significantly undersulfated [5]. No data are available regarding the bone phenotype of patients with disorders linked to the SLC26A2, most likely because clinical and research activities have focused mainly on the cartilage defects and on the molecular basis of reduced skeletal growth. In the current study we used growing (1 month-old) and young adult (2 month-old) dtd mice to investigate the effect of decreased Slc26a2 function on bone quality. Only male animals were considered in this study, since in the C57BL/6J background wild-type females age-related bone changes are more dramatic than in male littermates [22].

The bone mineral content and bone mineral density in dtd mice is reduced both at 1 and 2 months of age when compared to age-matched control animals, confirming the osteopenic phenotype observed at the histological level. Bone histomorphometry was performed to further characterize the bone phenotype and to find out whether it is a consequence of the cartilage defect or a primary bone defect. Static histomorphometry demonstrated that the trabecular and the cortical bone volume are significantly lower in dtd mice at the two age points considered compared to their wild-type littermates. As the cortical bone is partially formed by direct ossification without replacement of a cartilage anlage, the bone defect that we observe in our mouse model is not the consequence of a cartilage defect, but it is a primary bone defect. Indeed in 1-month old dtd mice, periosteal mineral apposition rate was decreased as well as cortical thickness, suggesting an impaired activity of individual periosteal osteoblasts.

The changes we observed on bone architecture suggest a defect in bone formation only in growing mice. Indeed, bone diameter was decreased as well as cortical and trabecular thickness in 1 month-old mice, but this was restored in young adult mice. In contrast, bone resorption appeared to be increased in both growing and adult mutant mice, since trabecular separation was enlarged.

Our in vivo and ex vivo analyses of bone cells demonstrated that bone cell differentiation was not impaired in dtd growing mice. Considering bone formation, the phenotype was different in growing and adult mice. We observed no changes in the extent of osteoblasts and labeled surfaces on trabecular, endosteal, and periosteal surfaces in 1 month-old dtd mice, suggesting that an identical number of actively bone forming osteoblasts is present in wild-type and mutant mice during growth. Accordingly, calvarial osteoblast from dtd mice proliferate normally, mineralize and express the same level of alkaline phosphatase as wild-type calvarial osteoblasts ex vivo. By contrast in young adult mice we observed an increased number of active osteoblasts at the endosteal and periosteal surfaces. This increase in bone formation rate induced a restoration of some of the architectural parameters in mutant mice, such as cortical thickness and trabecular thickness, but could not restore a normal bone diameter.

Secondary hyperparathyroidism, consistent with a similar bone phenotype, was excluded on the basis of normal levels of Ca2 + and PTH in mutant mouse sera.

In summary, the low trabecular bone volume observed in dtd mice was associated with a decreased individual osteoblastic activity during growth, since the mineral appositional rate as well as the osteoid thickness were lower in 1 month-old mutant mice compared to wt mice. These parameters returned to normal values at 2 months of age, therefore suggesting that the osteoblast defective activity was transient in dtd mice.

Considering bone resorption, we observed no changes in osteoclast surfaces in vivo in mutant mice. Moreover, ex vivo studies showed that dtd mice osteoclasts are able to differentiate as well as wild-type cells. However high bone resorption at the tissue level was demonstrated not only by the changes in bone architecture but also by the elevated level of two different biochemical markers of bone resorption in dtd mice. This can be explained only by an increased resorbing activity of individual osteoclasts.

In order to explain the reduced osteoblast activity and the high bone resorption despite an unchanged number of cells in mutant mice, we reasoned that chondroitin sulfate proteoglycan undersulfation in the bone of dtd mice could lead to altered bone matrix quality, making the bone extracellular matrix more easily degradable by osteoclasts and slowing down matrix synthesis by osteoblasts. Supporting this, we observed by electron microscopy that collagen fibrils had a smaller diameter and were less organized in the bone matrix of mutant mice compared to wild-types, suggesting that bone chondroitin sulfate proteoglycan undersulfation might affect bone extracellular matrix organization in dtd mice.

Supporting our hypothesis, it has previously been reported in literature that a disturbed assembly of the type II collagen fibrils in the cartilage matrix of growing mice can affect bone development, leading to increased bone resorption and alterations in the tissue properties [16]. Moreover, a deficiency in bone extracellular matrix quality caused by an altered diameter and irregular spacing of the collagen fibrils has been previously observed in the biglycan knock-out mouse [28,29].

In summary we have characterized the osteopenic phenotype of growing and young adult dtd mice reproducing human DTD resulting from A386V substitution in the Slc26a2. We excluded that bone alterations could be linked to defect in cell differentiation since osteoblast and osteoclast numbers and surfaces were normal in vivo, and their differentiation ex vivo was also normal. The altered bone extracellular matrix quality observed in dtd mice was associated with high osteoclast resorption and reduced osteoblast activity at the cell level. We therefore demonstrated that the SLC26A2 defect plays an indirect role in bone remodeling through proteoglycan undersulfation, suggesting that alterations in this ubiquitously expressed gene affect not only chondrogenesis, but also bone homeostasis, resulting in decreased bone mass and compromised bone architecture in dtd mice.

Acknowledgments

This work was supported by grants from MIUR (grant no. 20094C2H2M), Telethon-Italy (grant no. GGP06076), and the European Community (FP6, “EuroGrow” project, LSHM-CT-2007-037471).

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