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Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.

Ruptured abdominal aortic aneurysms (AAA) are associated with overall mortality rates up to 90%. Despite extensive research, mechanisms leading to AAA formation and advancement are still poorly understood. Smooth muscle cells (SMC) are predominant in the aortic medial layer and maintain the wall structure. Apoptosis of SMC is a well-known phenomenon in the pathophysiology of AAA. However, remaining SMC function is less extensively studied. The aim of this study is to assess the in vitro contractility of human AAA and non-pathologic aortic SMC. Biopsies were perioperatively harvested from AAA patients (n = 21) and controls (n = 6) and clinical data were collected. Contractility was measured using Electric Cell-substrate Impedance Sensing (ECIS) upon ionomycin stimulation. Additionally, SMC of 23% (5 out of 21) of AAA patients showed impaired maximum contraction compared to controls. Also, SMC from patients who underwent open repair after earlier endovascular repair and SMC from current smokers showed decreased maximum contraction vs. controls (p = 0.050 and p = 0.030, respectively). Our application of ECIS can be used to study contractility in other vascular diseases. Finally, our study provides with first proof that impaired SMC contractility might play a role in AAA pathophysiology.

Osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) are inherited connective tissue disorders caused by diverse genetic defects, many of which affect collagen biosynthesis. However, the identified genetic variants do not always fully explain the clinical heterogeneity observed in patients, highlighting the need for advanced models and imaging techniques to assess collagen structure and fibroblast behavior at the microscopic level. In this study, we employed 5-week three-dimensional (3D) dermal fibroblast cultures derived from patients with haploinsufficient (HI) and dominant-negative (DN) OI, EDS, and healthy controls. Using label-free higher harmonic generation microscopy (HHGM), we visualized and quantified secreted collagen fibers and fibroblast morphology in situ. We analyzed fibroblast 3D orientation, collagen fiber diameter, collagen amount per cell, and the spatial alignment between fibroblasts and collagen fibers. HI OI fibroblasts secreted significantly less collagen than both control and EDS-derived cells, while EDS samples exhibited thinner collagen fibers compared to controls. Across all groups, collagen fiber orientation was strongly correlated with fibroblast alignment, in line with the role of fibroblasts in matrix organization. In healthy controls and HI OI samples, we observed a depth-dependent, counterclockwise rotation in fibroblast orientation from the culture bottom to the surface-a pattern that was less prominent in DN OI and EDS samples, potentially reflecting altered matrix guidance in diseased tissues. Overall, the quantity and quality of collagen, as well as fibroblast morphology and organization, were markedly altered in the OI and EDS model systems. These alterations may mirror tissue-level manifestations of the diseases, demonstrating the physiological relevance of patient-derived 3D fibroblast models for OI and EDS, as well as the power of harmonic generation microscopy in probing the cellular and extracellular consequences of disease-related gene defects in collagen or its biosynthetic pathways. Extensions of this methodological approach provide a way towards deeper understanding of tissue-level manifestations of collagen dysregulation in connective tissue disorders.

A paper published in Orphanet Journal of Rare Diseases proposes a new classification of osteogenesis imperfecta (OI) based upon underlying pathological mechanisms. The proposed numbering of OI types conflicts with the currently used numbering and is likely to lead to confusion. In addition, classification of OI according to underlying pathogenic mechanisms is not novel.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic bone disorder, leading to progressive immobilization through the formation of bone in muscles, tendons, and ligaments. A variant in the gene results in a constitutively overactive ALK2 receptor, leading to the aberrant activation of the SMAD1/5/9 pathway. This activation occurs not only in response to Activin A, which does not normally activate this pathway, but also through heightened sensitivity to BMP ligands and even in the absence of ligand binding. This dysregulated signaling ultimately drives the formation of heterotopic ossification. The inhibition of the altered ALK2 receptor holds promise as a potential treatment strategy that is currently being investigated in several trials. In this study, we performed an in vitro characterization of novel kinase inhibitor KTI-2338 with high selectivity for the ALK2 receptor. Dermal human FOP and control fibroblasts were cultured in osteogenic medium with and without the inhibitor to assess the effect on transdifferentiation into osteoblast-like cells. Compound KTI-2338 elicited effects consistent with inhibiting aberrant Activin A signaling and receptor sensitization, through reductions in osteogenic markers and pSMAD1/5/9 expression levels. In line with this, a pattern of reduced Alizarin Red staining was observed following treatment with the compound, indicating reduced mineralization. These findings indicate that kinase inhibitor KTI-2338 disrupts the pathological processes underlying FOP and may offer a new therapeutic option for this devastating disease.

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells.

INTRODUCTION:Classical-like Ehlers-Danlos syndrome type 1 (clEDS1) is a very rare form of Ehlers-Danlos syndrome caused by tenascin-X deficiency, with only 56 individuals reported in medical literature. Tenascin-X is an extracellular matrix protein needed for collagen stability. Previous publications propose that individuals with clEDS1 might be at risk of gastrointestinal (GI) tract perforations and/or tracheal ruptures. The aim of this study was to characterize complications resulting from perforations of the GI tract and/or tracheal rupture in an international case series of individuals with clEDS1 due to disease-related tissue fragility.METHODS:This case series includes individuals with confirmed clEDS1 and GI perforations and/or tracheal ruptures from participating centers. Researchers who previously reported such individuals were contacted for additional information. A retrospective assessment of clinical features was performed.RESULTS:Fifteen individuals were included. Ten had spontaneous GI perforations, 7 of whom had multiple GI perforations. Almost all had severe diverticulosis. Three individuals experienced iatrogenic tracheal ruptures.DISCUSSION:Severe GI complications, such as perforation, and tracheal rupture were observed in a substantial number of individuals with clEDS1. As these features seem significantly more common in clEDS1 than in the average population, we advise vigilance during intubation and GI endoscopic interventions of individuals with clEDS1. Routine referrals to clinical geneticists are recommended for patients with symptoms indicative of clEDS1, especially with unexplained GI perforations and connective tissue symptoms. Our findings offer valuable insights for the clinical management of clEDS1 and underscore the importance of specialized care, providing a foundation for improved clinical guidelines and preventive strategies.

Plastin-3 (PLS3) encodes T-plastin, an actin-bundling protein mediating the formation of actin filaments by which numerous cellular processes are regulated. Loss-of-function genetic defects in PLS3 are reported to cause X-linked osteoporosis and childhood-onset fractures. However, the molecular etiology of PLS3 remains elusive. Functional compensation by actin-bundling proteins ACTN1, ACTN4, and FSCN1 was investigated in zebrafish following morpholino-mediated pls3 knockdown. Primary dermal fibroblasts from six patients with a PLS3 variant were also used to examine expression of these proteins during osteogenic differentiation. In addition, Pls3 knockdown in the murine MLO-Y4 cell line was employed to provide insights in global gene expression. Our results showed that ACTN1 and ACTN4 can rescue the skeletal deformities in zebrafish after pls3 knockdown, but this was inadequate for FSCN1. Patients’ fibroblasts showed the same osteogenic transdifferentiation ability as healthy donors. RNA-seq results showed differential expression in Wnt1, Nos1ap, and Myh3 after Pls3 knockdown in MLO-Y4 cells, which were also associated with the Wnt and Th17 cell differentiation pathways. Moreover, WNT2 was significantly increased in patient osteoblast-like cells compared to healthy donors. Altogether, our findings in different bone cell types indicate that the mechanism of PLS3-related pathology extends beyond actin-bundling proteins, implicating broader pathways of bone metabolism.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that causes bone fragility due to pathogenic variants in genes responsible for the synthesis of type I collagen. Efforts to classify the high clinical variability in OI led to the Sillence classification. However, this classification only partially takes into account extraskeletal manifestations and the high genetic variability. Little is known about the relation between genetic variants and phenotype as of yet. The aim of the study was to create a clinically relevant genetic stratification of a cohort of 675 Dutch OI patients based on their pathogenic variant types and to provide an overview of their respective medical care demands. The clinical records of 675 OI patients were extracted from the Amsterdam UMC Genome Database and matched with the records from Statistics Netherlands (CBS). The patients were categorized based on their harbored pathogenic variant. The information on hospital admissions, outpatient clinic visits, medication, and diagnosis-treatment combinations (DTCs) was compared between the variant groups. OI patients in the Netherlands appear to have a higher number of DTCs, outpatient clinic visits, and hospital admissions when compared to the general Dutch population. Furthermore, medication usage seems higher in the OI cohort in comparison to the general population. The patients with a COL1A1 or COL1A2 dominant negative missense non-glycine substitution appear to have a lower health care need compared to the other groups, and even lower than patients with COL1A1 or COL1A2 haploinsufficiency. It would be useful to include the variant type in addition to the Sillence classification when categorizing a patient’s phenotype.

Osteocytes sense mechanical loads and transduce mechanical signals into a chemical response. They are the most abundant bone cells deeply embedded in mineralized bone matrix, which affects their regulatory activity in the mechanical adaptation of bone. The specific location in the calcified bone matrix hinders studies on osteocytes in the in vivo setting. Recently, we developed a three‐dimensional mechanical loading model of human osteocytes in their native matrix, allowing to study osteocyte mechanoresponsive target gene expression in vitro. Here we aimed to identify differentially expressed genes by mapping the response of human primary osteocytes in their native matrix to mechanical loading using RNA sequencing. Human fibular bone was retrieved from 10 donors (age: 32–82 years, 5 female, 5 male). Cortical bone explants (8.0 × 3.0 × 1.5 mm; length × width × height) were either not loaded or mechanically loaded by 2000 or 8000 μɛ for 5 minutes, followed by 0, 6, or 24 hours post‐culture without loading. High‐quality RNA was isolated, and differential gene expression analysis performed by R2 platform. Real‐time PCR was used to confirm differentially expressed genes. Twenty‐eight genes were differentially expressed between unloaded and loaded (2000 or 8000 μɛ) bone at 6 hours post‐culture, and 19 genes at 24 hours post‐culture. Eleven of these genes were related to bone metabolism, ie, EGR1, FAF1, H3F3B, PAN2, RNF213, SAMD4A, and TBC1D24 at 6 hours post‐culture, and EGFEM1P, HOXD4, SNORD91B, and SNX9 at 24 hours post‐culture. Mechanical loading significantly decreased RNF213 gene expression, which was confirmed by real‐time PCR. In conclusion, mechanically loaded osteocytes differentially expressed 47 genes, of which 11 genes were related to bone metabolism. RNF213 might play a role in mechanical adaptation of bone by regulating angiogenesis, which is a prerequisite for successful bone formation. The functional aspects of the differentially expressed genes in bone mechanical adaptation requires future investigation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.