Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

Secreted factors from synovial fibroblasts immediately regulate gene expression in articular chondrocytes.

Although articular cartilage degeneration in osteoarthritis represents a major public health problem, there is still no molecular approach to prevent this pathology by blocking specific molecules. We have previously applied genome-wide expression analyses with porcine samples to identify specific markers of either growth plate or articular cartilage. Since the molecular differences were also found in cultured chondrocytes derived from both sites, we utilized primary porcine articular chondrocytes (PPACs) for the present study and analyzed, if and how they respond to synoviocyte-derived molecules. PPACs were treated by conditioned medium from porcine synovial fibroblasts (SF-CM) for 2, 6 and 24 h. Gene expression was subsequently monitored by qRT-PCR and microarray analysis. We found that short-term administration of SF-CM to PPACs significantly reduced expression of chondrocyte markers, while it induced expression of SDC4, encoding syndecan-4, a positive regulator of articular cartilage breakdown. Consistently, expression of MMP3, a putative downstream effector of syndecan-4 was strongly induced by SF-CM in PPACs. We identified an MMP3-inducing fraction in the range of 40 kDa after gel filtration, and we confirmed our findings in three-dimensional PPAC cultures, where SF-CM also reduced the glycosaminoglycan content. Taken together, our data suggest that synovial fibroblasts secrete one or more molecule(s) that activate specific signaling events in articular chondrocytes. Identifying a responsible ligand receptor pair(s) might pave the way to develop molecular therapies to reduce the severity of osteoarthritis.

Th17 cell frequency is associated with low bone mass in primary sclerosing cholangitis.

BACKGROUND & AIMS: Osteoporotic fractures are a major cause of morbidity and reduced quality of life in patients with primary sclerosing cholangitis (PSC), a progressive bile duct disease of unknown origin. Although it is generally assumed that this pathology is a consequence of impaired calcium homeostasis and malabsorption, the cellular and molecular causes of PSC-associated osteoporosis are unknown. METHODS: We determined bone mineral density by dual-X-ray absorptiometry and assessed bone microstructure by high-resolution peripheral quantitative computed tomography in patients with PSC. Laboratory markers of liver and bone metabolism were measured, and liver stiffness was assessed by FibroScan. We determined the frequency of Th17 cells by the ex vivo stimulation of peripheral blood mononuclear cells in a subgroup of 40 patients with PSC. To investigate the potential involvement of IL-17 in PSC-associated bone loss, we analyzed the skeletal phenotype of mice lacking Abcb4 and/or Il-17. RESULTS: Unlike in patients with primary biliary cholangitis, bone loss in patients with PSC was not associated with disease duration or liver fibrosis. However, we observed a significant negative correlation between the bone resorption biomarker deoxypyridinoline and bone mineral density in the PSC cohort, indicating increased bone resorption. Importantly, the frequency of Th17 cells in peripheral blood was positively correlated with the urinary deoxypyridinoline level and negatively correlated with bone mass. We observed that Abcb4-deficient mice displayed a low-bone-mass phenotype, which was corrected by an additional Il-17 deficiency or anti-IL-17 treatment, whereas the liver pathology was unaffected. CONCLUSIONS: Our findings demonstrate that an increased frequency of Th17 cells is associated with bone resorption in PSC. Whether antibody-based IL-17 blockade is beneficial against bone loss in patients with PSC should be addressed in future studies. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease characterized by progressive bile duct destruction. One serious complication of PSC is reduced bone mass resulting in increased fracture risk. Herein, we demonstrate that Th17 cells mediate bone loss in PSC by inducing bone resorption, which suggests that antibody-based IL-17 blockade might be beneficial for the treatment of bone loss in affected patients.

The Lysosomal Protein Arylsulfatase B Is a Key Enzyme Involved in Skeletal Turnover.

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate-resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6-phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS-VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5-/- mice, Arsb-deficient mice display lysosomal storage accumulation in osteoclasts, impaired osteoclast activity, and high trabecular bone mass. Of note, the most prominent lysosomal storage accumulation was observed in osteocytes from Arsb-deficient mice, yet this pathology did not impair production of sclerostin (Sost) and Fgf23. Because the influence of enzyme replacement therapy (ERT) on bone remodeling in MPS-VI is still unknown, we additionally treated Arsb-deficient mice by weekly injection of recombinant human ARSB from 12 to 24 weeks of age. We found that the high bone mass phenotype of Arsb-deficient mice and the underlying bone cell deficits were fully corrected by ERT in the trabecular compartment. Taken together, our results do not only show that the function of Acp5 in osteoclasts is linked to dephosphorylation and activation of lysosomal enzymes, they also provide an important proof-of-principle for the feasibility of ERT to correct bone cell pathologies in lysosomal storage disorders. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Early bone tissue aging in human auditory ossicles is accompanied by excessive hypermineralization, osteocyte death and micropetrosis.

Within the mineralized bone, osteocytes form a multifunctional mechanosensitive network orchestrating bone remodelling. A preserved osteocyte population is a crucial determinant of bone quality. In human auditory ossicles, the early decrease in osteocyte numbers but maintained integrity remains an unexplained phenomenon that might serve for sound transmission from air to the labyrinth. Here we analysed the frequency, size and composition of osteocyte lacunae in the auditory ossicles of 22 individuals from early postnatal period to old age. Mineralization of the bone matrix was determined using backscattered electron imaging. No signs of bone remodelling were observed above the age of 1 year. We detected characteristics of early bone tissue aging, such as decrease in osteocytes, lower total lacunar density and lacunar area, as well as high matrix mineralization accompanied by distinct accumulation of micropetrotic lacunae and decreased indentation depths. The majority of these changes took place in the first months and years of life, while afterwards only minor reorganization was present. With osteocyte apoptosis potentially being a consequence of low mechanical stimuli, the early loss of osteocytes without initiation of bone remodelling indicates an adaptive response conserving the architecture of the auditory ossicles and ensuring stable sound transmission throughout life.

High Bone Turnover in Mice Carrying a Pathogenic Notch2 Mutation Causing Hajdu-Cheney Syndrome.

Hajdu-Cheney syndrome (HCS) is a rare autosomal-dominant disorder primarily characterized by acro-osteolysis and early-onset osteoporosis. Genetically, HCS is caused by nonsense or deletion mutations within exon 34 of the NOTCH2 gene, resulting in premature translational termination and production of C-terminally truncated NOTCH2 proteins that are predicted to activate NOTCH2-dependent signaling. To understand the role of Notch2 in bone remodeling, we developed a mouse model of HCS by introducing a pathogenic mutation (6272delT) into the murine Notch2 gene. By µCT and undecalcified histology, we observed generalized osteopenia in two independent mouse lines derived by injection of different targeted embryonic stem (ES) cell clones, yet acro-osteolysis did not occur until the age of 52 weeks. Cellular and dynamic histomorphometry revealed a high bone turnover situation in Notch2+/HCS mice, since osteoblast and osteoclast indices were significantly increased compared with wild-type littermates. Whereas ex vivo cultures failed to uncover cell-autonomous gain-of-functions within the osteoclast or osteoblast lineage, an unbiased RNA sequencing approach identified Tnfsf11 and Il6 as Notch-signaling target genes in bone marrow cells cultured under osteogenic conditions. Because we further observed that the high-turnover pathology of Notch2+/HCS mice was fully normalized by alendronate treatment, our results demonstrate that mutational activation of Notch2 does not directly control osteoblast activity but favors a pro-osteoclastic gene expression pattern, which in turn triggers high bone turnover. © 2017 American Society for Bone and Mineral Research.

Comparison of contact radiographed and stained histological sections for osseointegration analysis of dental implants: an in vivo study.

OBJECTIVES: Histology is still regarded as the gold-standard to determine bone implant contact (BIC) as a parameter representing implant stability. As the further processing of cut slices for contact radiography (CR) to stained and polished histological sections is time consuming and error prone, our aim was to assess agreement between CR and Giemsa-eosin (GE) stained sections with regard to dental implants. STUDY DESIGN: Threaded dental titanium implants (n = 54) from the maxillae of Goettingen minipigs were evaluated. After 28 and 56 days, BIC and the ratio of bone volume to total volume (BV/TV; 1000 µm) were determined on the same sections by using CR and GE staining, and the results were compared. RESULTS: Moderate differences for BIC (0.6%; P = .53) and BV/TV (1.3%; P = .01) between the methods were determined, in which CR overestimated BIC and BV/TV. A strong correlation was seen between the modalities concerning BIC (28 days: r = 0.84; 56 days: r = 0.85; total: r = 0.85) and BV/TV (r = 0.96; r = 0.94; r = 0.96; all: P < .0001). CONCLUSIONS: CR enabled determination of the bone-to-implant interface in comparison with GE-stained sections. BIC and BV/TV were slightly overestimated but correlated strongly between the methods. Therefore, if BIC and BV/TV are sufficient endpoints, CR is adequate and no further preparation and staining are necessary.

Severe bone loss and multiple fractures in SCN8A-related epileptic encephalopathy.

Mutations in the SCN8A gene encoding the neuronal voltage-gated sodium channel Nav1.6 are known to be associated with epileptic encephalopathy type 13. We identified a novel de novo SCN8A mutation (p.Phe360Ala, c.1078_1079delTTinsGC, Exon 9) in a 6-year-old girl with epileptic encephalopathy accompanied by severe juvenile osteoporosis and multiple skeletal fractures, similar to three previous case reports. Skeletal assessment using dual energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT) and serum analyses revealed a combined trabecular and cortical bone loss syndrome with elevated bone resorption. Likewise, when we analyzed the skeletal phenotype of 2week-old Scn8a-deficient mice we observed reduced trabecular and cortical bone mass, as well as increased osteoclast indices by histomorphometric quantification. Based on this cumulative evidence the patient was treated with neridronate (2mg/kg body weight administered every 3months), which fully prevented additional skeletal fractures for the next 25months. Taken together, our data provide evidence for a negative impact of SCN8A mutations on bone mass, which can be positively influenced by anti-resorptive treatment.

Differential effects of Calca-derived peptides in male mice with diet-induced obesity.

Key metabolic hormones, such as insulin, leptin, and adiponectin, have been studied extensively in obesity, however the pathophysiologic relevance of the calcitonin family of peptides remains unclear. This family includes calcitonin (CT), its precursor procalcitonin (PCT), and alpha calcitonin-gene related peptide (αCGRP), which are all encoded by the gene Calca. Here, we studied the role of Calca-derived peptides in diet-induced obesity (DIO) by challenging Calcr-/- (encoding the calcitonin receptor, CTR), Calca-/-, and αCGRP-/- mice and their respective littermates with high-fat diet (HFD) feeding for 16 weeks. HFD-induced pathologies were assessed by glucose tolerance, plasma cytokine and lipid markers, expression studies and histology. We found that DIO in mice lacking the CTR resulted in impaired glucose tolerance, features of enhanced nonalcoholic steatohepatitis (NASH) and adipose tissue inflammation compared to wildtype littermates. Furthermore, CTR-deficient mice were characterized by dyslipidemia and elevated HDL levels. In contrast, mice lacking Calca were protected from DIO, NASH and adipose tissue inflammation, and displayed improved glucose tolerance. Mice exclusively lacking αCGRP displayed a significantly less improved DIO phenotype compared to Calca-deficient mice. In summary, we demonstrate that the CT/CTR axis is involved in regulating plasma cholesterol levels while Calca, presumably through PCT, seems to have a detrimental effect in the context of metabolic disease. Our study provides the first comparative analyses of the roles of Calca-derived peptides and the CTR in metabolic disease.

Vitamin D regulates osteocyte survival and perilacunar remodeling in human and murine bone.

Osteocytes are the most abundant bone cells and are highly regulated by external stimuli. Vitamin D and osteocytes cooperatively regulate bone remodeling as well as phosphate and calcium homeostasis. However, it is unclear if vitamin D regulates osteocyte number, connectivity or size in the setting of altered bone formation or impaired mineralization. Sixty iliac crest biopsies of patients with varying vitamin D levels were examined to analyze osteocyte number, osteocyte connectivity and osteocyte viability using high-resolution imaging. Osteocyte parameters were also quantified in mice lacking the vitamin D receptor (Vdr-/-) and in wildtype littermates. The cortical and cancellous bone of patients with vitamin D deficiency exhibited a significant decrease in the number of viable osteocytes, as well as increased osteocyte apoptosis and impaired osteocyte connectivity, based on evaluation of the canalicular network. The number of osteocytes was also decreased in Vdr-deficient mice, in comparison to wildtype controls, and this was accompanied by enlargement of osteocyte lacunae. A high calcium diet normalized the osteocyte lacunar area in Vdr-deficient mice, but failed to normalize osteocyte number. Thus, a diet-independent decrease in osteocyte number in Vdr-deficient mice suggests a mechanism that is directly dependent on the VDR, since vitamin D may promote the transition from osteoblasts to osteocytes. The increase in lacunar area the in Vdr-deficient mice, which is normalized by the high calcium diet suggests this phenotype is due to osteocytic osteolysis. These investigations demonstrate that vitamin D plays a role in the regulation of osteocyte number and perilacunar remodeling.

Comparative 3D micro-CT and 2D histomorphometry analysis of dental implant osseointegration in the maxilla of minipigs.

AIM: The bone implant contact (BIC) has traditionally been evaluated with histological methods. Thereupon, strong correlations of two-dimensional (2D) BIC have been detected between µCT and destructive histology. However, due to the high intra-sample variability in BIC values, one histological slice is not sufficient to represent 3D BIC. Therefore, our aim has been to correlate the averaged values of 3-4 histological sections to 3D µCT. MATERIAL AND METHODS: Fifty-four implants inserted into the maxilla of 14 minipigs were evaluated. Two different time points were selected to assess the 3D BIC (distance to implant: 2-5 voxels), an inner ring (6-30 voxels) and an outer ring (55-100 voxels) using µCT (voxel size: 10 µm) and to correlate the values to histomorphometry. RESULTS: Strong correlations (p < 0.0001; 28 days, 56 days, total) were seen between µCT and histomorphometry concerning BIC (r = 0.84, r = 0.85, r = 0.83), the inner ring (r = 0.87, r = 0.87, r = 0.88) and the outer ring (r = 0.85, r = 0.85, r = 0.88). Closer to the implant, µCT values were higher compared with histomorphometry. CONCLUSION: Although 3-4 histological slices per implant seem to predict the 3D BIC, µCT might be advantageous because of its non-destructive 3D character. The healing time may not impact on the comparability.

How the European eel (Anguilla anguilla) loses its skeletal framework across lifetime.

European eels (Anguilla anguilla) undertake an impressive 5 000 km long migration from European fresh waters through the North Atlantic Ocean to the Sargasso Sea. Along with sexual maturation, the eel skeleton undergoes a remarkable morphological transformation during migration, where a hitherto completely obscure bone loss phenomenon occurs. To unravel mechanisms of the maturation-related decay of the skeleton, we performed a multiscale assessment of eels' bones at different life-cycle stages. Accordingly, the skeleton reflects extensive bone loss that is mediated via multinucleated bone-resorbing osteoclasts, while other resorption mechanisms such as osteocytic osteolysis or matrix demineralization were not observed. Preserving mechanical stability and releasing minerals for energy metabolism are two mutually exclusive functions of the skeleton that are orchestrated in eels through the presence of two spatially segregated hard tissues: cellular bone and acellular notochord. The cellular bone serves as a source of mineral release following osteoclastic resorption, whereas the mineralized notochord sheath, which is inaccessible for resorption processes due to an unmineralized cover layer, ensures sufficient mechanical stability as a part of the notochord sheath. Clearly, an eel's skeleton is structurally optimized to meet the metabolic challenge of fasting and simultaneous sexual development during an exhausting journey to spawning areas, while the function of the vertebral column is maintained to achieve this goal.

Chronic skin inflammation leads to bone loss by IL-17-mediated inhibition of Wnt signaling in osteoblasts.

Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.

Osteoblast-specific Notch2 inactivation causes increased trabecular bone mass at specific sites of the appendicular skeleton.

Notch signaling is a key pathway controlling various cell fate decisions during embryogenesis and adult life. It is activated by binding of specific ligands to four different Notch receptors that are subsequently cleaved by presenilins to release an intracellular domain that enters the nucleus and activates specific transcription factors. While the skeletal analysis of various mouse models with activated or inactivated Notch signaling has demonstrated a general impact of this pathway on bone remodeling, the more recent identification of NOTCH2 mutations in individuals with Hajdu-Cheney syndrome (HCS) has highlighted its human relevance. Since HCS is primarily characterized by skeletal defects, these latter findings led us to analyze the specific role of Notch2 in skeletal remodeling. After observing Notch2 expression in osteoblasts and osteoclasts, we utilized Runx2-Cre and Lyz2-Cre mice to inactivate Notch2 in cells of the osteoblast or osteoclast lineage, respectively. Whereas Notch2(fl/fl)/Lyz2-Cre mice did not display significant alterations of skeletal growth, bone mass or remodeling, Notch2(fl/fl)/Runx2-Cre mice progressively developed skeletal abnormalities in long bones. More specifically, these mice displayed a striking increase of trabecular bone mass in the proximal femur and the distal tibia at 6 and 12months of age. Whereas undecalcified sectioning of the respective regions did not reveal impaired osteocyte differentiation as a potential trigger for the observed phenotype, ex vivo experiments with bone marrow cells identified an increased osteogenic capacity of Notch2(fl/fl)/Runx2-Cre cultures. Collectively, our findings demonstrate that Notch2 physiologically regulates bone remodeling by inhibiting trabecular bone formation in the appendicular skeleton. Understanding the underlying mechanisms may help to improve diagnosis and therapy of HCS.

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition, but Causes a Transient Reduction of Articular Cartilage Thickness.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements.

INTRODUCTION: Calcium silicate cements (CSCs) with the addition of nanohydroxyapatite and calcium carbonate play a critical role in dental applications. To further improve their properties, particularly radiopacity and biointeractivity, the fluoride-containing radiopacifier ytterbium trifluoride (YbF3) was added to their composition, and biological and mechanical characteristics were evaluated. METHODS: YbF3 was added to 3 different CSCs: cement I (CSC + calcium carbonate), cement II (CSC + nanohydroxyapatite), and Portland cement. Material characterization encompassed measurements of pH, calcium, ytterbium, and fluoride ion release; radiopacity; setting time; porosity; microindentation properties; wettability; and Fourier transform infrared spectroscopic, x-ray diffraction, and scanning electron microscopic analyses. Osteoblast- and osteoclast-like cells were grown on the materials' surface to evaluate their adherence. RESULTS: The addition of calcium carbonate, nanohydroxyapatite, and 30 wt% of YbF3 improved radiopacity and the setting time of experimental cements. The pH values did not differ among the groups. The greatest ytterbium and fluoride releases occurred in the Portland cement + YbF3 group. Combined x-ray diffraction and Fourier transform infrared spectroscopic analysis showed the presence of calcium hydroxide and calcium silicate hydrates. In addition, the presence of calcium ytterbium fluoride and ytterbium oxide proved that YbF3 reacted with cement compounds. Wettability of cement I + YbF3 was superior to other formulations, but its porosity and microindentation properties were weaker than in the Portland cement + YbF3 mixture. Cement II + YbF3 presented micromechanical indentation and porosity characteristics similar to the Portland-based cement formulation. Osteoclast- and osteoblast-like cells adhered to the cements' surfaces without alteration of the cell structural integrity. CONCLUSIONS: YbF3-containing CSCs with nanostructured hydroxyapatite and calcium carbonate are well suited for dental application.

Sharpin Controls Osteogenic Differentiation of Mesenchymal Bone Marrow Cells.

The cytosolic protein Sharpin is a component of the linear ubiquitin chain assembly complex, which regulates NF-κB signaling in response to specific ligands, such as TNF-α. Its inactivating mutation in chronic proliferative dermatitis mutation (Cpdm) mice causes multiorgan inflammation, yet this phenotype is not transferable into wild-type mice by hematopoietic stem cell transfer. Recent evidence demonstrated that Cpdm mice additionally display low bone mass, and that this osteopenia is corrected by Tnf deletion. Because the cellular mechanism underlying this pathology, however, was still undefined, we performed a thorough skeletal phenotyping of Cpdm mice on the basis of nondecalcified histology and cellular and dynamic histomorphometry. We show that the trabecular and cortical osteopenia in Cpdm mice is solely explained by impaired bone formation, whereas osteoclastogenesis is unaffected. Consistently, Cpdm primary calvarial cells display reduced osteogenic capacity ex vivo, and the same was observed with CD11b(-) bone marrow cells. Unexpectedly, short-term treatment of these cultures with TNF-α did not reveal an impaired molecular response in the absence of Sharpin. Instead, genome-wide and gene-specific expression analyses revealed that Cpdm mesenchymal cells display increased responsiveness toward TNF-α-induced expression of specific cytokines, such as CXCL5, IL-1ß, and IL-6. Therefore, our data not only demonstrate that the skeletal defects of Cpdm mice are specifically caused by impaired differentiation of osteoprogenitor cells, they also suggest that increased cytokine expression in mesenchymal bone marrow cells contributes to the inflammatory phenotype of Cpdm mice.

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

The Anti-Osteoanabolic Function of Sclerostin Is Blunted in Mice Carrying a High Bone Mass Mutation of Lrp5.

Activating mutations of the putative Wnt co-receptor Lrp5 or inactivating mutations of the secreted molecule Sclerostin cause excessive bone formation in mice and humans. Previous studies have suggested that Sclerostin functions as an Lrp5 antagonist, yet clear in vivo evidence was still missing, and alternative mechanisms have been discussed. Moreover, because osteoblast-specific inactivation of ß-catenin, the major intracellular mediator of canonical Wnt signaling, primarily affected bone resorption, it remained questionable, whether Sclerostin truly acts as a Wnt signaling antagonist by interacting with Lrp5. In an attempt to address this relevant question, we generated a mouse model (Col1a1-Sost) with transgenic overexpression of Sclerostin under the control of a 2.3-kb Col1a1 promoter fragment. These mice displayed the expected low bone mass phenotype as a consequence of reduced bone formation. The Col1a1-Sost mice were then crossed with two mouse lines carrying different high bone mass mutations of Lrp5 (Lrp5(A170V) and Lrp5(G213V)), both of them potentially interfering with Sclerostin binding. Using µCT-scanning and histomorphometry we found that the anti-osteoanabolic influence of Sclerostin overexpression was not observed in Lrp5(A213V/A213V) mice and strongly reduced in Lrp5(A170V/A170V) mice. As a control we applied the same strategy with mice overexpressing the transmembrane Wnt signaling antagonist Krm2 and found that the anti-osteoanabolic influence of the Col1a1-Krm2 transgene was not affected by either of the Lrp5 mutations. Taken together, our data support the concept that Sclerostin inhibits bone formation through Lrp5 interaction, yet their physiological relevance remains to be established.

3D-tracking the regenerative potential of the mandible with micro-CTs.

PURPOSE: The treatment of large bone defects is a challenging problem especially when the mandible is affected. Bone healing is dependent on the defect size and the integrity of periosteum. So far, these both aspects have not been investigated separately. The aim of this study was to evaluate the healing potential of the mandibular bone with the help of three-dimensional micro-computed tomography (CT). METHODS: The angle of the mandible was exposed in 15 Wistar rats. A 3-mm core of bone was removed with a trephine. The local periosteum next to the defect was excised. Animals were randomized in five groups, which were ended 5, 10, 15, 28 and 56 days after operation. The mandible was excised and underwent micro-CT. For statistical evaluation, t-test statistics and regression analysis were applied. RESULTS: Characteristics of the defects began to change on the tenth postoperative day. Fifteen days until 4 weeks after intervention new mineralization processes could be observed. New bone grew from the borders into the defect. In the 2D study, bone apposition changed significantly from the beginning to week 8 (0.08 to 0.74 mm) as well as the 3D bone gain (0.05 % to 29.67 %) in t-test statistical evaluation. For development of the bone volume inside the defect linear as well as exponential regression analysis revealed a statistically significant connection. CONCLUSIONS: This study quantified the amount of newly grown bone during osseous regeneration. We could show that the mandible itself provides regenerative capacity without any intact periosteum.