Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) ...and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521(+/-) mice was rescued in Zfp521(+/-):Ebf1(+/-) mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis.
Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that ...drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper.
In oncogenic osteomalacia, the causative tumor is almost always difficult to find. A novel diagnostic approach is presented that facilitates a precise and rapid localization of the associated lesion ...by PET‐CT co‐registration using the radiotracer 68Ga‐DOTANOC.
In oncogenic osteomalacia, the causative tumor is almost always difficult to find. A novel diagnostic approach is presented that facilitates a precise and rapid localization of the associated lesion by PET‐CT co‐registration using the radiotracer 68Ga‐DOTANOC.
Introduction: Oncogenic osteomalacia (OOM) is an uncommon disorder characterized by hyperphosphaturia, hypophosphatemia, decreased vitamin D3 serum levels, and osteomalacia. The paraneoplastic syndrome is exclusively driven by a small somatostatin receptor (sst)‐positive tumor that produces phosphatonins, proteins that cause renal phosphate loss. OOM can be cured completely on tumor removal. However, the exact tumor localization is the most challenging step, because the lesion is notoriously difficult to detect by common imaging techniques.
Materials and Methods: A 60‐year‐old woman complained of severe pain in her back and chest wall, muscle weakness, and reduced physical activity for >1 year. She suffered a metatarsal fracture and presented with hyperphosphaturia and hypophosphatemia. OOM was suspected, and a meticulous search for the tumor was initiated by conventional imaging techniques, sst‐mediated imaging using 111In‐octreotide scintigraphy, and 68Ga‐DOTANOC‐based positron emission tomography (PET)‐CT co‐registration. 68Ga‐DOTANOC is a novel radiopharmaceutical compound in which the somatostatin analog octreotide is modified at position 3, chelated with DOTA, and complexed with 68Gallium. 68Ga‐DOTANOC has an improved affinity to sst2 and sst5 relative to other radiopeptides.
Results: Whereas common imaging techniques such as CT failed to localize the tumor, 111In‐octreotide scintigraphy was able to detect the lesion, but only PET‐CT using 68Ga‐DOTANOC revealed the exact tumor localization in the right femoral head. On tumor resection, the well being of the patient improved significantly, and biochemical parameters returned to normal.
Conclusions: 68Ga‐DOTANOC‐based PET‐CT is a novel and powerful approach to detect sst‐positive tumors in a timely manner and to provide highly resolved images facilitating the development of a therapeutic strategy.
Exploration of anabolic pathways in osteoblasts revealed that Zfp521, a 30‐zinc finger protein, is highly expressed at the periphery of mesenchymal condensations and in developing bones. In these ...structures it is expressed in chondroblasts, prehypertrophic chondrocytes, the periosteum, osteoblasts, osteoblast precursors, and osteocytes. Forced expression of Zfp521 in osteoblasts in vivo increases bone formation and bone mass, whereas preliminary data suggest that germline deletion leads to osteopenia. In contrast, overexpressing Zfp521 in vitro antagonizes, and knockdown favors, osteoblast differentiation and nodule formation. Zfp521 expression is inhibited by bone morphogenetic protein‐2 and stimulated by parathyroid hormone‐related protein. Mechanistically, Zfp521 binds to Runx2, repressing its transcriptional activity. These data support the hypothesis that Zfp521 both opposes the progression of precursors and promotes the maturation and function of mature osteoblasts. The balance between Zfp521 and Runx2 may therefore contribute to the regulation of osteoblast differentiation and bone formation.
Mechanical loading is crucial for bone remodeling and osteoblast differentiation. FosB belongs to the AP-1 family of transcription factors, a group of proteins known to regulate osteoblast ...differentiation and bone formation. In mice, FosB is rapidly induced by mechanical stress at the transcriptional level. The aim of this study was to determine the effect of different mechanical stretch patterns on FosB gene expression and on osteogenic differentiation of human osteoblast precursor cells. Human bone-marrow-derived mesenchymal precursor cells were grown in flexible silicone dishes and stimulated by a daily application of three rounds of 2
h of cyclic stretch of either 2% or 8% elongation at 1
Hz on 3 consecutive days using a special motor-driven apparatus. By real-time PCR, we quantified FosB mRNA and the expression of genes involved in osteoblast differentiation such as Runx2 and collagen 1 to determine the osteogenic effect of mechanical stretch. Stretching induced FosB transcription and the expression of osteoblast markers in partly committed human mesenchymal precursor cells in a stretch- and time-dependent manner. We conclude that cyclic stretch-induced FosB expression and the upregulation of osteoblast genes plays a role in osteogenic differentiation of human mesenchymal precursor cells.
In 1990 we advanced the hypothesis that frightened and frightening (FR) parental behavior would prove to be linked to both unresolved (U) adult attachment status as identified in the Adult Attachment ...Interview and to infant disorganized/disoriented (D) attachment as assessed in the Ainsworth Strange Situation. Here, we present a coding system for identifying and scoring the intensity of the three primary forms of FR behavior (frightened, threatening, and dissociative) as well as three subsidiary forms. We review why each primary form may induce fear of the parent (the infant's primary "haven of safety"), placing the infant in a disorganizing approach-flight paradox. We suggest that, being linked to the parent's own unintegrated traumatic experiences (often loss or maltreatment), FR behaviors themselves are often guided by parental fright, and parallel the three "classic" mammalian responses to fright: flight, attack, and freezing behavior. Recent studies of U to FR, as well as FR to D relations are presented, including findings regarding AMBIANCE/FR+. Links between dissociation, FR, U, and D are explored. Parallel processing and working memory are discussed as they relate to these phenomena.
Purpose It was our aim to establish an animal model and to investigate the tendon graft–to–bone and physis healing process in skeletally immature sheep after reconstruction of the anterior cruciate ...ligament (ACL). Methods Thirty-two immature sheep aged 4 months underwent a fully transphyseal ACL reconstruction by use of a soft-tissue graft. The animals were subsequently killed after 3, 6, 12, and 24 weeks and analyzed histologically and biomechanically. Results There was a transient hypertrophy of the physis tissue at the passing site of the graft. Anchoring Sharpey-like fibers evolved as early as 3 weeks after surgery. A strong expression of collagen III messenger ribonucleic acid within the first 6 weeks preceded this anchoring process. The maximum load to failure of the tendon graft in the reconstructed knees initially decreased to 37.8 ± 17.8 N after 3 weeks and was restored to 522.9 ± 113 N after 24 weeks. Tendon graft stiffness was restored to 86% when compared with the control knees. Conclusions The early anchoring by Sharpey fibers was found at 3 weeks with continued maturation to 24 weeks. This development of anchoring fibers corresponded to that of biomechanical strength, starting with 5% of the normal knee at 3 weeks and then 15.2% at 6 weeks, 41.2% at 12 weeks, and 69% at 24 weeks. Tendon graft–to–bone and physis healing in skeletally immature sheep is further characterized by a transient hypertrophy of the physis cartilage. The physis recovers well from the trauma of drilling and placement of a soft-tissue graft. The early development of Sharpey-like fibers results in a solid integration of the graft into bone in a timely manner. Clinical Relevance ACL reconstruction in skeletally immature individuals is still controversial. This study describes in detail the histologic and biomechanical stages of tendon graft healing to the bone and physis. These data enrich the existing knowledge of previous studies in adult sheep and may provide a basis for further research in the controversial field of ACL reconstruction during growth.
Purpose The aim of this study was to analyze the relation between bone mineral density (BMD) and femoral tunnel enlargement (TE) in a previously validated sheep model of soft-tissue anterior cruciate ...ligament (ACL) reconstruction. Methods Thirty sheep underwent ACL reconstruction by use of a soft-tissue graft at the age of 4 months. Graft fixation was achieved with the EndoButton (Smith & Nephew Endoscopy, Andover, MA) and Suture Washer (Smith & Nephew Endoscopy). Six animals were killed at 0, 3, 6, 12, and 24 weeks postoperatively. Each ACL-reconstructed knee was examined both by computed tomography to analyze the bone tunnel cross-sectional area and by dual-energy x-ray absorptiometry to analyze BMD. Results There was a significant increase in tunnel cross-sectional area. BMD decreased significantly within the first 3 weeks after surgery and increased thereafter. A positive correlation between TE and BMD was found. However, a subgroup analysis showed that there is no influence of BMD on the development of a tunnel widening. Conclusions The hypothesis that a TE would be associated with a loss in BMD was not confirmed. Tunnel widening during the first 6 months after ACL reconstruction is not affected by the transient changes in BMD. Clinical Relevance There is no correlation between TE and BMD in an experimental sheep model of ACL reconstruction. Translational investigations will determine whether this is also true in humans.
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