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Since our discovery in 2013 that genetic defects in PLS3 lead to bone fragility, the mechanistic details of this process have remained obscure. It has been established that PLS3 variants cause syndromic and nonsyndromic osteoporosis as well as osteoarthritis. PLS3 codes for an actin-bundling protein with a broad pattern of expression. As such, it is puzzling how PLS3 specifically leads to bone-related disease presentation. Our review aims to summarize the current state of knowledge regarding the function of PLS3 in the predominant cell types in the bone tissue, the osteocytes, osteoblasts and osteoclasts. This is related to the role of PLS3 in regulating mechanotransduction, calcium regulation, vesicle trafficking, cell differentiation and mineralization as part of the complex bone pathology presented by PLS3 defects. Considering the consequences of PLS3 defects on multiple aspects of bone tissue metabolism, our review motivates the study of its mechanism in bone diseases which can potentially help in the design of suitable therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhong, Pathak, Liang, Zhytnik, Pals, Eekhoff, Bravenboer and Micha.)

Introduction: Osteogenesis Imperfecta is a rare genetic connective tissue disorder, characterized by skeletal dysplasia and fragile bones. Currently only two mouse models have been reported for haploinsufficient (HI) mild Osteogenesis Imperfecta (OI); the Col1a1 +/Mov13 (Mov13) and the Col1a1 +/-365 mouse model. The Mov13 mice were created by random insertion of the Mouse Moloney leukemia virus in the first intron of the Col1a1 gene, preventing the initiation of transcription. Since the development of the Mov13 mice almost four decades ago and its basic phenotypic characterization in the 90s, there have not been many further studies. We aimed to extensively characterize the Mov13 mouse model in order to critically evaluate its possible use for preclinical studies of HI OI., Methods: Bone tissue from ten heterozygous Mov13 and ten wild-type littermates (WT) C57BL/6J mice (50% males per group) was analyzed at eight weeks of age with bone histomorphometry, micro computed tomography (microCT), 3-point bending, gene expression of different collagens, as well as serum markers of bone turnover., Results: The Mov13 mouse presented a lower bone strength and impaired material properties based on our results of 3-point bending and microCT analysis respectively. In contrast, no significant differences were found for all histomorphometric parameters. In addition, no significant differences in Col1a1 bone expression were present, but there was a significant lower P1NP concentration, a bone formation marker, measured in serum. Furthermore, bone tissue of Mov13 mice presented significantly higher expression of collagens ( Col1a2 , Col5a1 and Col5a2 ), and bone metabolism markers ( Bglap , Fgf23 , Smad7 , Edn1 and Eln ) compared to WT. Finally, we measured a significantly lower Col1a1 expression in heart and skin tissue and also determined a higher expression of other collagens in the heart tissue., Conclusion: Although we did not detect a significant reduction in Col1a1 expression in the bone tissue, a change in bone structure and reduction in bone strength was noted. Regrettably, the variability of the bone phenotype and the appearance of severe lymphoma in adult Mov13 mice, does not favor their use for the testing of new long-term drug studies. As such, a new HI OI type 1 mouse model is urgently needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Claeys, Zhytnik, Wisse, van Essen, Eekhoff, Pals, Bravenboer and Micha.)

Bone marrow adipose tissue (BMAT) is a dynamic tissue which is associated with osteoporosis, bone metastasis, and primary bone tumors. The aim of this study is to determine region-specific variations and age- and gender-specific differences in BMAT and BMAT composition in healthy subjects. In this cross-sectional study, we included 40 healthy subjects (26 male: mean age 49 years, range 22-75 years; 14 female: mean age 50 years, range 29-71) and determined the bone marrow signal fat fraction and bone marrow unsaturation in the spine (C3-L5), pelvis, femora, and tibiae using chemical shift encoding-based water-fat imaging (WFI) with multiple gradient echoes (mGRE). Regions of interest covered the individual vertebral bodies, pelvis and proximal epimetaphysis, diaphysis, and distal epimetaphysis of the femur and tibia. The spinal fat fraction increased from cervical to lumbar vertebral bodies (mean fat fraction ( ± SD or (IQR): cervical spine 0.37 ± 0.1; thoracic spine 0.41 ± 0.08. lumbar spine 0.46 ± 0.01; p < 0.001). The femoral fat fraction increased from proximal to distal (proximal 0.78 ± 0.09; diaphysis 0.86 (0.15); distal 0.93 ± 0.02; p < 0.001), while within the tibia the fat fraction decreased from proximal to distal (proximal 0.92 ± 0.01; diaphysis 0.91 (0.02); distal 0.90 ± 0.01; p < 0.001). In female subjects, age was associated with fat fraction in the spine, pelvis, and proximal femur (ρ = 0.88 p < 0.001; ρ = 0.87 p < 0.001; ρ = 0.63 p = 0.02; ρ = 0.74 p = 0.002, respectively), while in male subjects age was only associated with spinal fat fraction (ρ = 0.40 p = 0.04). Fat fraction and unsaturation were negatively associated within the spine (r = -0.40 p = 0.01), while in the extremities fat fraction and unsaturation were positively associated (distal femur: r = 0.42 p = 0.01; proximal tibia: r = 0.47, p = 0.002; distal tibia: r = 0.35 p = 0.03), both independent of age and gender. In conclusion, we confirm the distinct, age- and gender-dependent, distribution of BMAT throughout the human skeleton and we show that, contradicting previous animal studies, bone marrow unsaturation in human subjects is highest within the axial skeleton compared to the appendicular skeleton. Furthermore, we show that BMAT unsaturation was negatively correlated with BMAT within the spine, while in the appendicular skeleton, BMAT and BMAT unsaturation were positively associated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Beekman, Regenboog, Nederveen, Bravenboer, den Heijer, Bisschop, Hollak, Akkerman and Maas.)

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Ruiter, Smilde, Pals, Bravenboer, Knaus, Schoenmaker, Botman, Sánchez-Duffhues, Pacifici, Pignolo, Shore, van Egmond, Van Oosterwyck, Kaplan, Hsiao, Yu, Bocciardi, De Cunto, Longo Ribeiro Delai, de Vries, Hilderbrandt, Jaspers, Keen, Koolwijk, Morhart, Netelenbos, Rustemeyer, Scott, Stockklausner, ten Dijke, Triffit, Ventura, Ravazzolo, Micha and Eekhoff.)

In the field of rare bone diseases in particular, a broad care team of specialists embedded in multidisciplinary clinical and research environment is essential to generate new therapeutic solutions and approaches to care. Collaboration among clinical and research departments within a University Medical Center is often difficult to establish, and may be hindered by competition and non-equivalent cooperation inherent in a hierarchical structure. Here we describe the "collaborative organizational model" of the Amsterdam Bone Center (ABC), which emerged from and benefited the rare bone disease team. This team is often confronted with pathologically complex and under-investigated diseases. We describe the benefits of this model that still guarantees the autonomy of each team member, but combines and focuses our collective expertise on a clear shared goal, enabling us to capture synergistic and innovative opportunities for the patient, while avoiding self-interest and possible harmful competition., (Copyright © 2020 Eekhoff, Micha, Forouzanfar, de Vries, Netelenbos, Klein-Nulend, van Loon, Lubbers, Schwarte, Schober, Raijmakers, Teunissen, de Graaf, Lammertsma, Yaqub, Botman, Treurniet, Smilde, Bökenkamp, Boonstra, Kamp, Nieuwenhuijzen, Visser, Baayen, Dahele, Eeckhout, Goderie, Smits, Gilijamse, Karagozoglu, van de Valk, Dickhoff, Moll, Verbraak, Curro-Tafili, Ghyczy, Rustemeyer, Saeed, Maugeri, Pals, Ridwan-Pramana, Pekel, Schoenmaker, Lems, Winters, Botman, Giannakópoulos, Koolwijk, Janssen, Kloen, Bravenboer, Smit and Helder.)

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts., (Copyright © 2020 Schoenmaker, Botman, Sariyildiz, Micha, Netelenbos, Bravenboer, Kelder, Eekhoff and De Vries.)

Osteocytes are the most abundant (~95%) cells in bone with the longest half-life (~25 years) in humans. In the past osteocytes have been regarded as vestigial cells in bone, since they are buried inside the tough bone matrix. However, during the last 30 years it has become clear that osteocytes are as important as bone forming osteoblasts and bone resorbing osteoclasts in maintaining bone homeostasis. The osteocyte cell body and dendritic processes reside in bone in a complex lacuno-canalicular system, which allows the direct networking of osteocytes to their neighboring osteocytes, osteoblasts, osteoclasts, bone marrow, blood vessels, and nerves. Mechanosensing of osteocytes translates the applied mechanical force on bone to cellular signaling and regulation of bone adaptation. The osteocyte lacuno-canalicular system is highly efficient in transferring external mechanical force on bone to the osteocyte cell body and dendritic processes via displacement of fluid in the lacuno-canalicular space. Osteocyte mechanotransduction regulates the formation and function of the osteoblasts and osteoclasts to maintain bone homeostasis. Osteocytes produce a variety of proteins and signaling molecules such as sclerostin, cathepsin K, Wnts, DKK1, DMP1, IGF1, and RANKL/OPG to regulate osteoblast and osteoclast activity. Various genetic abnormality-associated rare bone diseases are related to disrupted osteocyte functions, including sclerosteosis, van Buchem disease, hypophosphatemic rickets, and WNT1 and plastin3 mutation-related disorders. Meticulous studies during the last 15 years on disrupted osteocyte function in rare bone diseases guided for the development of various novel therapeutic agents to treat bone diseases. Studies on genetic, molecular, and cellular mechanisms of sclerosteosis and van Buchem disease revealed a role for sclerostin in bone homeostasis, which led to the development of the sclerostin antibody to treat osteoporosis and other bone degenerative diseases. The mechanism of many other rare bone diseases and the role of the osteocyte in the development of such conditions still needs to be investigated. In this review, we mainly discuss the knowledge obtained during the last 30 years on the role of the osteocyte in rare bone diseases. We speculate about future research directions to develop novel therapeutic drugs targeting osteocyte functions to treat both common and rare bone diseases., (Copyright © 2020 Pathak, Bravenboer and Klein-Nulend.)

Bone tissue degeneration is an urgent clinical issue, making it a subject of intensive research. Chronic skeletal disease forms can be prevalent, such as the age-related osteoporosis, or rare, in the form of monogenetic bone disorders. A barrier in the understanding of the underlying pathological process is the lack of accessibility to relevant material. For this reason, cells of non-bone tissue are emerging as a suitable alternative for models of bone biology. Fibroblasts are highly suitable for this application; they populate accessible anatomical locations, such as the skin tissue. Reports suggesting their utility in preclinical models for the study of skeletal diseases are increasingly becoming available. The majority of these are based on the generation of an intermediate stem cell type, the induced pluripotent stem cells, which are subsequently directed to the osteogenic cell lineage. This intermediate stage is circumvented in transdifferentiation, the process regulating the direct conversion of fibroblasts to osteogenic cells, which is currently not well-explored. With this mini review, we aimed to give an overview of existing osteogenic transdifferentiation models and to inform about their applications in bone biology models., (Copyright © 2020 Claeys, Bravenboer, Eekhoff and Micha.)

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt -/- , Kit W/W-v ) and development of specific BMA deletion models would be highly desirable for this purpose., (Copyright © 2020 Tratwal, Labella, Bravenboer, Kerckhofs, Douni, Scheller, Badr, Karampinos, Beck-Cormier, Palmisano, Poloni, Moreno-Aliaga, Fretz, Rodeheffer, Boroumand, Rosen, Horowitz, van der Eerden, Veldhuis-Vlug and Naveiras.)

Research into bone marrow adiposity (BMA) has expanded greatly since the late 1990s, leading to development of new methods for the study of bone marrow adipocytes. Simultaneously, research fields interested in BMA have diversified substantially. This increasing interest is revealing fundamental new knowledge of BMA; however, it has also led to a highly variable nomenclature that makes it difficult to interpret and compare results from different studies. A consensus on BMA nomenclature has therefore become indispensable. This article addresses this critical need for standardised terminology and consistent reporting of parameters related to BMA research. The International Bone Marrow Adiposity Society (BMAS) was formed in 2017 to consolidate the growing scientific community interested in BMA. To address the BMA nomenclature challenge, BMAS members from diverse fields established a working group (WG). Based on their broad expertise, the WG first reviewed the existing, unsystematic nomenclature and identified terms, and concepts requiring further discussion. They thereby identified and defined 8 broad concepts and methods central to BMA research. Notably, these had been described using 519 unique combinations of term, abbreviation and unit, many of which were overlapping or redundant. On this foundation a second consensus was reached, with each term classified as " to use " or " not to use ." As a result, the WG reached a consensus to craft recommendations for 26 terms related to concepts and methods in BMA research. This was approved by the Scientific Board and Executive Board of BMAS and is the basis for the present recommendations for a formal BMA nomenclature. As an example, several terms or abbreviations have been used to represent "bone marrow adipocytes," including BMAds, BM-As , and BMAs . The WG decided that BMA should refer to "bone marrow adiposity"; that BM-A is too similar to BMA ; and noted that "Ad" has previously been recommended to refer to adipocytes. Thus, it was recommended to use BMAds to represent bone marrow adipocytes . In conclusion, the standard nomenclature proposed in this article should be followed for all communications of results related to BMA. This will allow for better interactions both inside and outside of this emerging scientific community., (Copyright © 2020 Bravenboer, Bredella, Chauveau, Corsi, Douni, Ferris, Riminucci, Robey, Rojas-Sutterlin, Rosen, Schulz and Cawthorn.)