Mouse Model Of Osteogenesis Imperfecta Research Articles

Galunisertib downregulates mutant type I collagen expression and promotes MSCs osteogenesis in pediatric osteogenesis imperfecta

Qualitative alterations in type I collagen due to pathogenic variants in the COL1A1 or COL1A2 genes, result in moderate and severe Osteogenesis Imperfecta (OI), a rare disease characterized by bone fragility. The TGF-β signaling pathway is overactive in OI patients and certain OI mouse models, and inhibition of TGF-β through anti-TGF-β monoclonal antibody therapy in phase I clinical trials in OI adults is rendering encouraging results. However, the impact of TGF-β inhibition on osteogenic differentiation of mesenchymal stem cells from OI patients (OI-MSCs) is unknown. The following study demonstrates that pediatric skeletal OI-MSCs have imbalanced osteogenesis favoring the osteogenic commitment. Galunisertib, a small molecule inhibitor (SMI) that targets the TGF-β receptor I (TβRI), favored the final osteogenic maturation of OI-MSCs. Mechanistically, galunisertib downregulated type I collagen expression in OI-MSCs, with greater impact on mutant type I collagen, and concomitantly, modulated the expression of unfolded protein response (UPR) and autophagy markers. In vivo, galunisertib improved trabecular bone parameters only in female oim/oim mice. These results further suggest that type I collagen is a tunable target within the bone ECM that deserves investigation and that the SMI, galunisertib, is a promising new candidate for the anti-TGF-β targeting for the treatment of OI.

Extra-Skeletal Manifestations in Osteogenesis Imperfecta Mouse Models.

Osteogenesis imperfecta (OI) is a rare heritable connective tissue disorder of skeletal fragility with an incidence of roughly 1:15,000. Approximately 85% of the pathogenic variants responsible for OI are in the type I collagen genes, COL1A1 and COL1A2, with the remaining pathogenic OI variants spanning at least 20 additional genetic loci that often involve type I collagen post-translational modification, folding, and intracellular transport as well as matrix incorporation and mineralization. In addition to being the most abundant collagen in the body, type I collagen is an important structural and extracellular matrix signaling molecule in multiple organ systems and tissues. Thus, OI disease-causing variants result not only in skeletal fragility, decreased bone mineral density (BMD), kyphoscoliosis, and short stature, but can also result in hearing loss, dentinogenesis imperfecta, blue gray sclera, cardiopulmonary abnormalities, and muscle weakness. The extensive genetic and clinical heterogeneity in OI has necessitated the generation of multiple mouse models, the growing awareness of non-skeletal organ and tissue involvement, and OI being more broadly recognized as a type I collagenopathy.This has driven the investigation of mutation-specific skeletal and extra-skeletal manifestations and broadened the search of potential mechanistic therapeutic strategies. The purpose of this review is to outline several of the extra-skeletal manifestations that have recently been characterized through the use of genetically and phenotypically heterogeneous mouse models of osteogenesis imperfecta, demonstrating the significant potential impact of OI disease-causing variants as a collagenopathy (affecting multiple organ systems and tissues), and its implications to overall health.

Combining anabolic loading and raloxifene improves bone quantity and some quality measures in a mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) increases fracture risk due to changes in bone quantity and quality caused by mutations in collagen and its processing proteins. Current therapeutics improve bone quantity, but do not treat the underlying quality deficiencies. Male and female G610C+/− mice, a murine model of OI, were treated with a combination of raloxifene and in vivo axial tibial compressive loading starting at 10 weeks of age and continuing for 6 weeks to improve bone quantity and quality. Bone geometry and mechanical properties were measured to determine whole bone and tissue-level material properties. A colocalized Raman/nanoindentation system was used to measure chemical composition and nanomechanical properties in newly formed bone compared to old bone to determine if bone formed during the treatment regimen differed in quality compared to bone formed prior to treatment. Lastly, lacunar geometry and osteocyte apoptosis were assessed. OI mice were able to build bone in response to the loading, but this response was less robust than in control mice. Raloxifene improved some bone material properties in female but not male OI mice. Raloxifene did not alter nanomechanical properties, but loading did. Lacunar geometry was largely unchanged with raloxifene and loading. However, osteocyte apoptosis was increased with loading in raloxifene treated female mice. Overall, combination treatment with raloxifene and loading resulted in positive but subtle changes to bone quality.

Collagen mutation and age contribute to differential craniofacial phenotypes in mouse models of osteogenesis imperfecta.

Craniofacial and dentoalveolar abnormalities are present in all types of osteogenesis imperfecta (OI). Mouse models of the disorder are critical to understand these abnormalities and underlying OI pathogenesis. Previous studies on severely affected OI mice report a broad spectrum of craniofacial phenotypes, exhibiting some similarities to the human disorder. The Brtl/+ and G610c/+ are moderately severe and mild-type IV OI, respectively. Little is known about the aging effects on the craniofacial bones of these models and their homology to human OI. This study aimed to analyze the Brtl/+ and G610c/+ craniofacial morphometries during aging to establish suitability for further OI craniofacial bone intervention studies. We performed morphological measurements on the micro-CT-scanned heads of 3-wk-old, 3-mo-old, and 6-mo-old female Brtl/+ and G610c/+ mice. We observed that Brtl/+ skulls are shorter in length than WT (P < .05), whereas G610c/+ skulls are similar in length to their WT counterparts. The Brtl/+ mice exhibit alveolar bone with a porotic-like appearance that is not observed in G610c/+. As they age, Brtl/+ mice show severe bone resorption in both the maxilla and mandible (P < .05). By contrast, G610c/+ mice experience mandibular resorption consistently across all ages, but maxillary resorption is only evident at 6mo (P < .05). Western blot shows high osteoclastic activities in the Brtl/+ maxilla. Both models exhibit delayed pre-functional eruptions of the third molars (P < .05), which are similar to those observed in some bisphosphonate-treated OI subjects. Our study shows that the Brtl/+ and G610c/+ mice display clear features found in type IV OI patients; both show age-related changes in the craniofacial growth phenotype. Therefore, understanding the craniofacial features of these models and how they age will allow us to select the most accurate mouse model, mouse age, and bone structure for the specific craniofacial bone treatment of differing OI groups.

RNA-based bone histomorphometry: method and its application to explaining postpubertal bone gain in a G610C mouse model of osteogenesis imperfecta.

Bone histomorphometry is a well-established approach to assessing skeletal pathology, providing a standard evaluation of the cellular components, architecture, mineralization, and growth of bone tissue. However, it depends in part on the subjective interpretation of cellular morphology by an expert, which introduces bias. In addition, diseases like osteogenesis imperfecta (OI) and fibrous dysplasia are accompanied by changes in the morphology and function of skeletal tissue and cells, hindering consistent evaluation of some morphometric parameters and interpretation of the results. For instance, traditional histomorphometry combined with collagen turnover markers suggested that reduced bone formation in classical OI is accompanied by increased bone resorption. In contrast, the well-documented postpubertal reduction in fractures would be easier to explain by reduced bone resorption after puberty, highlighting the need for less ambiguous measurements. Here we propose an approach to histomorphometry based on in situ mRNA hybridization, which uses Col1a1 as osteoblast and Ctsk as osteoclast markers. This approach can be fully automated and eliminates subjective identification of bone surface cells. We validate these markers based on the expression of Bglap, Ibsp, and Acp5. Comparison with traditional histological and tartrate-resistant acid phosphatase staining of the same sections suggests that mRNA-based analysis is more reliable. Unlike inconclusive traditional histomorphometry of mice with α2(I)-Gly610 to Cys substitution in the collagen triple helix, mRNA-based measurements reveal reduced osteoclastogenesis in 11-wk-old animals consistent with the postpubertal catch-up osteogenesis observed by microCT. We optimize the technique for cryosections of mineralized bone and sections of paraffin-embedded decalcified tissue, simplifying and broadening its applications. We illustrate the application of the mRNA-based approach to human samples using the example of a McCune-Albright syndrome patient. By eliminating confounding effects of altered cellular morphology and the need for subjective morphological evaluation, this approach may provide a more reproducible and accessible evaluation of bone pathology.

THU455 Sclerostin Inhibitor Use In The Medical Management Of Osteogenesis Imperfecta Type III

Abstract Disclosure: M.D. Lundholm: None. A. Derkyi: None. S. Talvacchio: None. J.C. Marini: Other; Self; Ultragenyx, Amgen Inc. L.Z. Khan: None. Background: Osteogenesis imperfecta (OI) is a rare collagen-related hereditary skeletal disorder with an increased fracture risk from minimal trauma, short stature, and bone deformity. OI is associated with defects in collagen structure or synthesis, high bone turnover, and paradoxical bone hypermineralization. The optimal medical therapies for bone health in these patients are unknown. Clinical Case: A 27-year-old woman with OI presented to clinic for bone health. She was diagnosed with OI type III by phenotype, collagen biochemistry, and genetic analyses (heterozygous for a COL1A1 Gly187Ala substitution) following fractures at age 4 mos. She experienced dozens of fractures, has had orthopedic hardware in bilateral femora, left tibia, humerus, and radius, and right tibia, as well as spine fusion at age 7 yrs. She had not fractured in over 3 yrs which she attributed to the stability of her automatic wheelchair. She had no family history of OI and had been in studies at the NIH; from ages 6-9 yrs she received pamidronate (1 mg/kg/cycle q3m) with BMD improvement. Presently, her vitals were normal, with height of 3’6” (25-50th percentile for type III OI females) and weight of 64 lbs (BMI 25.5 kg/m2). On exam she had blue sclera, midface hypoplasia, dentinogenesis imperfecta, severe kyphosis and scoliosis, and a barrel chest. She had normal calcium, PTH, and 25-OH vitamin D levels. Skull CT showed 1 mm increase in basilar invagination (16 to 17 mm). DEXA demonstrated progressive decline in radial BMD from 0.671 g/cm2 to 0.625 g/cm2 over 3 yrs (z-score -0.4 to -1.1) into the osteopenic range. For worsening bone disease, she is starting romosozumab 210 mg monthly. Discussion: While several agents increase BMD in OI, there are no placebo-controlled studies to show an effect on fracture rate. Bisphosphonates including pamidronate are used most commonly, with increased areal BMD, decreased bone turnover, but also increased mineralization of already brittle bone. Overall, two Cochran reviews failed to support decreased fracture rates from bisphosphonates, and there is risk of adynamic bone with chronic use. There is even less evidence for use of denosumab which is additionally associated with a rebound effect upon discontinuation. Sclerostin inhibitors are the newest agents with both anabolic and antiresorptive effects and good results in OI mouse models. Phase 2 data with sestrusumab showed increased BMD and decreased bone turnover. Ongoing studies are looking at sclerostin inhibitor use in adults and adolescents. From a practical standpoint, romosozumab has a shorter effect with quicker offset than bisphosphonates if needed to stop for fracture healing. Conclusion: Sclerostin inhibitors are an option for the medical treatment of OI. Further investigation is needed to determine long-term safety, optimal duration, and use of sclerostin inhibitors in combination with antiresorptive agents in OI. Presentation: Thursday, June 15, 2023

Neurocranial growth in the OIM mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a disorder of type I collagen characterized by abnormal bone formation. The OI craniofacial phenotype includes midfacial underdevelopment, as well as neurocranial changes (e.g., macrocephaly and platybasia) that may also affect underlying nervous tissues. This study aims to better understand how OI affects the integrated development of the neurocranium and the brain. Juvenile and adult mice with OI (OIM) and unaffected wild type (WT) littermates were imaged using in vivo micro-computed tomography (microCT). Virtual endocast models were used to measure brain volume, and 3D landmarks were collected from the cranium and brain endocasts. Geometric morphometric analyses were used to compare brain shape and integration between the genotypes. OIM mice had increased brain volumes (relative to cranial centroid size) only at the juvenile stage. No significant difference was seen in cranial base angle (CBA) between OIM and WT mice. However, CBA was higher in juvenile than in adult OIM mice. Brain shape was significantly different between OIM and WT mice at both stages, with OIM mice having more globular brains than WT mice. Neurocranial and brain morphology were strongly integrated within both genotypes, while adult OIM mice tended to have lower levels of skull-brain integration than WT mice. These results suggest that neurocranial dysmorphologies in OI may be more severe at earlier stages of postnatal development. Decreased skull-brain integration in adult mice suggests that compensatory mechanisms may exist during postnatal growth to maintain neurological function despite significant changes in neurocranial morphology.

Irisin reduces bone fracture by facilitating osteogenesis and antagonizing TGF-β/Smad signaling in a growing mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a congenital disorder characterized by muscle defect and skeletal fragility, and no cure is yet available. Crosstalk between bone and muscle has become a new coming focus of therapeutic strategy in OI. Irisin, a secreted myokine, was found to be involved in regulating bone metabolism, and may be beneficial for the treatment of OI. However, its effects in OI have yet to be determined. This study sought to determine whether Irisin therapy is capable of reducing fracture risk in OI and to investigate the potential mechanisms of action. Fibronectin type III domain containing 5 (FNDC5)/Irisin expression was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. In vivo, X-ray was used for fracture counting and micro-CT, dynamic histomorphometry analysis, immunohistochemistry, histomorphometry, and biomechanical test were used to evaluate the effects of Irisin on fracture frequency and bone quality in OI mouse model, oim/oim mouse. In vitro, osteogenesis-related gene expressions were determined by quantitative real-time PCR (qRT-PCR), western blot, and osteoblastogenesis assay were assessed by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining. Mechanistically, cell immunofluorescence staining, co-immunoprecipitation (co-IP) (Co-IP), molecular docking, western blot, luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay were used for elucidating the mechanisms of how Irisin antagonized transforming growth factor-β (TGF-β)/Smad signaling in oim/oim osteoblasts and further attenuated the inhibitory effect of TGF-β1 on osteogenic differentiation. Musculoskeletal system-related FNDC5/Irisin was decreased in the serum, muscle, and bone in oim/oim mice. Irisin administration reduced bone fracture and attenuated bone abnormalities by improving bone mass and strength and facilitating the expression of osteogenic differentiation markers. In vivo study and in vitro experiments showed that Irisin antagonized TGF-β/Smad signaling by interfering with TGF-β1-TGF-β receptor II (TβRII) binding. In oim/oim osteoblasts, Irisin alleviated TGF-β1-induced suppression of osteogenic differentiation through both integrin-dependent and integrin-independent mechanisms. Independent of integrin receptors, Irisin affected osteogenesis by activating ERK/p38 signaling and counteracting TGF-β/Smad2/3 signaling. In particular, Irisin alleviated TGF-β1-induced inhibition of Runx2 function at the osteocalcin promoter through decreasing Smad2/3 signaling and inducing HADC4/5 degeneration. Collectively, Irisin could effectively reduce bone fracture in oim/oim mice through promoting osteogenesis and counteracting TGF-β/Smad signaling. Findings from this study provided evidence for using Irisin as a potential therapeutic reagent to prevent the progression of OI.

Distinct type I collagen alterations cause intrinsic lung and respiratory defects of variable severity in mouse models of osteogenesis imperfecta.

Type I collagen alterations cause osteogenesis imperfecta (OI), a connective tissue disorder characterized by severe bone fragility. Patients with OI can suffer from significant pulmonary manifestations including severe respiratory distress in the neonatal period and a progressive decline in respiratory function in adulthood. We and others have shown intrinsic lung defects in some mouse models of OI. In this large study, we performed histological, histomorphometric, microcomputed tomography and invasive studies on oim/+, Col1a2+/G610C , CrtapKO and oim/oim mice, mimicking mild to moderate to severe OI, with the overall goal of determining the extent of their pulmonary and respiratory mechanics defects and whether these defects correlate with the skeletal disease severity and affect each sex equally. Although with variable severity, OI lung histology consistently showed alveolar simplification with enlarged acinar airspace and reduced alveolar surface. Numerous respiratory mechanics parameters, including respiratory system resistance and elastance, tissue damping, inspiratory capacity, total lung capacity, and others, were significantly and similarly impacted in CrtapKO and oim/oim but not in oim/+ or Col1a2+/G610C compared to control mice. Our data indicate that the impact of type I collagen alterations and OI on lung morphology and function positively correlate with the severity of the extracellular matrix deficiency. Moreover, the respiratory defects were more pronounced in male compared to female mice. It will be important to determine whether our observations in mice translate to OI patients and to dissect the respective contribution of intrinsic lung defects vs. extrinsic skeletal defects to impaired lung function in OI. KEY POINTS: Different type I collagen alterations in mouse models of osteogenesis imperfecta (OI) cause similar abnormal lung histology, with alveolar simplification and reduced alveolar surface, reminiscent of emphysema. Several respiratory mechanics parameters are altered in mouse models of OI. The impact of type I collagen alterations and OI on lung morphology and function positively correlate with the severity of the extracellular matrix deficiency. Respiratory defects were more pronounced in male compared to female mice. It will be important to determine whether our observations in mice translate to OI patients and to dissect the respective contribution of intrinsic lung defects vs. extrinsic skeletal defects to impaired lung function in OI.

A multi-model approach to predict efficacious clinical dose for an anti-TGF-β antibody (GC2008) in the treatment of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited bone dysplasias characterized by reduced skeletal mass and bone fragility. Although the primary manifestation of the disease involves the skeleton, OI is a generalized connective tissue disorder that requires a multidisciplinary treatment approach. Recent studies indicate that application of a transforming growth factor beta (TGF-β) neutralizing antibody increased bone volume fraction (BVF) and strength in an OI mouse model and improved bone mineral density (BMD) in a small cohort of patients with OI. In this work, we have developed a multitiered quantitative pharmacology approach to predict human efficacious dose of a new anti-TGF-β antibody drug candidate (GC2008). This method aims to translate GC2008 pharmacokinetic/pharmacodynamic (PK/PD) relationship in patients, using a number of appropriate mathematical models and available preclinical and clinical data. Compartmental PK linked with an indirect PD effect model was used to characterize both pre-clinical and clinical PK/PD data and predict a GC2008 dose that would significantly increase BMD or BVF in patients with OI. Furthermore, a physiologically-based pharmacokinetic model incorporating GC2008 and the body's physiological properties was developed and used to predict a GC2008 dose that would decrease the TGF-β level in bone to that of healthy individuals. By using multiple models, we aim to reveal information for different aspects of OI disease that will ultimately lead to a more informed dose projection of GC2008 in humans. The different modeling efforts predicted a similar range of pharmacologically relevant doses in patients with OI providing an informed approach for an early clinical dose setting.

Skeletal muscle mitochondrial function and whole-body metabolic energetics in the +/G610C mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is rare heritable connective tissue disorder that most often arises from mutations in the type I collagen genes, COL1A1 and COL1A2, displaying a range of symptoms including skeletal fragility, short stature, blue-gray sclera, and muscle weakness. Recent investigations into the intrinsic muscle weakness have demonstrated reduced contractile generating force in some murine models consistent with patient population studies, as well as alterations in whole body bioenergetics. Muscle weakness is found in approximately 80% of patients and has been equivocal in OI mouse models. Understanding the mechanism responsible for OI muscle weakness is crucial in building our knowledge of muscle bone cross-talk via mechanotransduction and biochemical signaling, and for potential novel therapeutic approaches. In this study we evaluated skeletal muscle mitochondrial function and whole-body bioenergetics in the heterozygous +/G610C (Amish) mouse modeling mild/moderate human type I/VI OI and minimal skeletal muscle weakness. Our analyses revealed several changes in the +/G610C mouse relative to their wildtype littermates including reduced state 3 mitochondrial respiration, increased mitochondrial citrate synthase activity, increased Parkin and p62 protein content, and an increased respiratory quotient. These changes may represent the ability of the +/G610C mouse to compensate for mitochondrial and metabolic changes that may arise due to type I collagen mutations and may also account for the lack of muscle weakness observed in the +/G610C model relative to the more severe OI models.

Modeling anabolic and antiresorptive therapies for fracture healing in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic bone fragility disorder that features frequent fractures. Bone healing outcomes are contingent on a proper balance between bone formation and resorption, and drugs such as bone morphogenetic proteins (BMPs) and bisphosphonates (BPs) have shown to have utility in modulating fracture repair. While BPs are used for OI to increase BMD and reduce pain and fracture rates, there is little evidence for using BMPs as local agents for fracture healing (alone or with BPs). In this study, we examined wild-type and OI mice (Col1a2+/G610C ) in a murine tibial open fracture model with (i) surgery only/no treatment, (ii) local BMP-2 (10 µg), or (iii) local BMP-2 and postoperative zoledronic acid (ZA; 0.1 mg/kg total dose). Microcomputed tomography reconstructions of healing fractures indicated BMP-2 was less effective in an OI setting, however,BMP-2 +ZA led to considerable increases in bone volume (+193% WT, p < 0.001; +154% OI, p < 0.001) and polar moment of inertia (+125% WT, p < 0.01; +248% OI, p < 0.05). Tissue histology revealed a thinning of the neocortex of the callus in BMP-2 treated OI bone, but considerable retention of woven bone in the healing callus with BMP + ZA specimens. These data suggest a cautious approach may be warranted with the sole application of BMP-2 in an OI surgical setting as a bone graft substitute. However, this may be overcome by off-label BPadministration.

Combination treatment with growth hormone and zoledronic acid in a mouse model of Osteogenesis imperfecta.

Osteogenesis imperfecta (OI) or brittle bone disease is a genetic disorder that results in bone fragility. Bisphosphonates such as zoledronic acid (ZA) are used clinically to increase bone mass and reduce fracture risk. Human growth hormone (hGH) has been used to promote long bone growth and forestall short stature in children with OI. The potential for hGH to improve bone quality, particularly in combination with ZA has not been robustly studied. A preclinical study was performed using n=80 mice split evenly by genotype (WT, Col1a2+/G610C). Groups of n=10 were treated with +/-ZA and +/-hGH in a factorial design for each genotype. Outcome measures included bone length, isolated muscle mass, bone parameters assessed by microCT analysis, dynamic histomorphometry, and biomechanical testing. Treatment with hGH alone led to an increase in femur length in WT but not OI mice, however bone length was increased in both genotypes with the combination of hGH/ZA. MicroCT showed that hGH/ZA treatment increased cortical BV in both WT (+15%) and OI mice (+14.3%); hGH/ZA were also found to be synergistic in promoting cortical thickness in OI bone. ZA was found to have a considerably greater positive impact on trabecular bone than hGH. ZA was found to suppress bone turnover, and this was rescued by hGH treatment in terms of cortical periosteal perimeter, but not by dynamic bone remodeling. Statistically significant improvements in long bone by microCT did not translate into improvements in mechanical strength in a 4-point bending test, nor did vertebral strength improve in L4 compression testing in WT/OI bone. These data support hGH/ZA combination as a treatment for short stature, however the improvements granted by hGH alone and in combination with ZA on bone quality are modest. Increased periosteal perimeter does show promise in improving bone strength in OI, however a longer treatment time may be required to see effects on bone strength through mechanical testing.

The effects of systemic and local factors on regulation of skeletal and fat tissues in a mouse model of osteogenesis imperfecta

The effects of systemic and local factors on regulation of skeletal and fat tissues in a mouse model of osteogenesis imperfecta

ER stress reduction by 4-PBA treatment improves bone phenotypes in the Aga2 mouse model of osteogenesis imperfecta

ER stress reduction by 4-PBA treatment improves bone phenotypes in the Aga2 mouse model of osteogenesis imperfecta

Neurocranial Growth in the OIM Mouse Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a disorder of type I collagen characterized by abnormal bone formation and weakened bone architecture. Human patients with OI have larger cranial vaults (macrocephaly), altered cranial base morphology including basilar invagination and platybasia (skull base flattening), and midfacial underdevelopment. The neurocranial changes may affect both underlying nervous tissue and growth patterns of the facial skeleton. However, we still do not fully understand how and when these divergent morphologies occur. The aims of this study are: (1) to investigate the integrated development of the skull and the brain in a mouse model of OI; and (2) to identify the developmental trajectories of these structures to facilitate future therapeutic interventions. We hypothesize that compared to unaffected mice, mice with OI will have decreased brain volumes due to an overall reduction in cranial size and decreased cranial base angles (CBA) due to platybasia.To test these hypotheses, we used the osteogenesis imperfecta murine (OIM or B6C3FE a/a‐Col1a2oim/J), a model for the severe type III OI in humans, and unaffected wild‐type (WT) littermates. Mice were imaged using in vivo micro‐computed tomography (micro‐CT) at the juvenile (week 4; 10 OIM/14 WT) and adult (week 16; 9 OIM/11 WT) stages. All measurements were taken in 3D Slicer software. 82 cranial landmarks were used to calculate centroid size, an estimate of overall head size. The segmentations tool was used to create virtual endocasts as a proxy for brain volume. The angle tool was used to measure CBA in the midsagittal plane using three landmarks: foramen cecum, midsphenoidal synchondrosis, and basion. Mann‐Whitney U tests were used to compare centroid sizes, brain volumes, and CBA between the genotypes.Both juvenile (p=0.008) and adult (p=0.003) OIM mice were found to have absolutely smaller brains than WT mice. However, OIM mice also have significantly smaller cranial centroid sizes compared to WT mice (p=0.003, p&lt;0.001). When scaled to cranial size, juvenile mice had relatively larger brain volumes (p=0.016) but adult OIM relative brain volumes were not significantly different from WT. No significant difference was seen in CBA at the juvenile (p=0.065) or adult (p=0.171) stages, however a trend was observed for decreased CBA at the adult stage.These results suggest that neurocranial dysmorphologies in OI may be more severe at earlier stages of postnatal development. Previous analyses of these mice have documented relative skeletal macrocephaly in both juvenile and adults, however here we document an increase in relative endocranial volume only at the juvenile stage. A reduction in CBA during growth, possibly due to platybasia, may underlie this decoupling between external and internal cranial morphology. Future work will investigate the effect of CBA on facial growth and midfacial underdevelopment in these mice. A better understanding of the integration and growth trajectory of the neurocranium is foundational for formulating treatments to manage basicranial instabilities in patients with OI.

Craniofacial Bone Mineral Density at Muscle Attachment Sites in Mice with Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic disease commonly caused by mutations in the COL1A1 or COL1A2 gene affecting the production of type I collagen, a major component of bone that determines the toughness of bone, or its resistance to fracture. Thus, patients with OI present with brittle bones and higher rates of skeletal fractures. Additionally, defects in the collagen framework of developing bones impair their biomineralization, further compromising bone mechanical properties. Previous work in our lab investigated BMD at cranial locations which experience compressive forces during feeding (e.g. joints). Muscles also contribute to cranial growth through tensile forces exerted on bone during contraction, but the relationship between muscle function and bone quality in OI is unclear. The aim of this study is to quantify the BMD in a mouse model of OI, with a focus on skeletal sites associated with cranial musculature. We hypothesize that the mice with OI will have lower BMD in skeletal areas associated with muscle attachment compared to their unaffected littermates.To test our hypothesis, we used the homozygous recessive OI murine (OIM) mouse (B6C3Fe a/a‐Col1a2oim/J), a model for the severe type III OI seen in humans. Adult (16 weeks old) OIM mice (n=11) and wildtype (WT, n=13) littermates were CT scanned, and BMD was measured using the Bruker CTAnalyzer software in 7 regions of interest encompassing muscle attachment sites on the mandible, zygomatic arch, and parietal bone. For each region, BMD values were compared between genotypes by a Kruskal‐Wallis test.OI mice had significantly lower BMD at the coronoid process (p=0.007), parietal bone (p=0.031), and posterior mylohyoid line (p=0.007) compared to WT mice. No significant differences were found in BMD at the gonial angle or zygomatic arch. This suggests a disproportionate weakness of the temporalis and mylohyoid muscles in mice with OI, but not in the masseter muscle. While type I collagen defects impact the structural organization of skeletal muscle, it is possible that masseter muscle function is preserved in OIM mice because of its key roles in rodent feeding and postural control of the jaw. The imbalance between temporalis and masseter muscles may contribute to the prevalence of dental malocclusions in human patients with type III OI.The findings of this study support further exploration of specific muscle groups (e.g. jaw adductors/temporalis, hyoid muscles/mylohyoid) and efforts to improve their functions during feeding, as a way of recovering facial growth and preventing dental malocclusions in OI. A better appreciation of muscle‐bone interactions in the skull and their effect on bone quality can potentially be used to inform oro‐motor therapies to improve feeding function, muscle strength, and facial bone quality for patients with OI.

Infrared Spectroscopy-Determined Bone Compositional Changes Associated with Anti-Resorptive Treatment of the oim/oim Mouse Model of Osteogenesis Imperfecta.

Applications of vibrational spectroscopy to assess bone disease and therapeutic interventions are continually advancing, with tissue mineral and protein composition frequently investigated. Here, we used two spectroscopic approaches for determining bone composition in a mouse model (oim) of the brittle bone disease osteogenesis imperfecta (OI) with and without antiresorptive agent treatment (alendronate, or ALN, and RANK-Fc). Near-infrared (NIR) spectral analysis using a fiber optic probe and attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) mode were applied to investigate bone composition, including water, mineral, and protein content. Spectral parameters revealed differences among the control wildtype (WT) and OIM groups. NIR spectral analysis of protein and water showed that OIM mouse humerii had ∼50% lower protein and ∼50% higher overall water content compared to WT bone. Moreover, some OIM-treated groups showed a reduction in bone water compared to OIM controls, approximating values observed in WT bone. Differences in bone quality based on increased mineral content and reduced carbonate content were also found between some groups of treated OIM and WT bone, but crystallinity did not differ among all groups. The spectroscopically determined parameters were evaluated for correlations with gold-standard mechanical testing values to gain insight into how composition influenced bone strength. As expected, bone mechanical strength parameters were consistently up to threefold greater in WT mice compared to OIM groups, except for stiffness in the ALN-treated OIM groups. Furthermore, bone stiffness, maximum load, and post-yield displacement showed the strongest correlations with NIR-determined protein content (positive correlations) and bound-water content (negative correlations). These results demonstrate that in this study, NIR spectral parameters were more sensitive to bone composition differences than ATR parameters, highlighting the potential of this nondestructive approach for screening of bone diseases and therapeutic efficacy in pre-clinical models.

Inhibition of TGF‐β Increases Bone Volume and Strength in a Mouse Model of Osteogenesis Imperfecta

ABSTRACTOsteogenesis imperfecta (OI), is a genetic disorder of bone fragility caused by mutations in collagen I or proteins involved in collagen processing. Previous studies in mice and human OI bones have shown that excessive activation of TGF‐β signaling plays an important role in dominant and recessive OI disease progression. Inhibition of TGF‐β signaling with a murine pan‐specific TGF‐β neutralizing antibody (1D11) was shown to significantly increase trabecular bone volume and long bone strength in mouse models of OI. To investigate the frequency of dosing and dose options of TGF‐β neutralizing antibody therapy, we assessed the effect of 1D11 on disease progression in a dominant OI mouse model (col1a2 gene mutation at G610C). In comparison with OI mice treated with a control antibody, we attempted to define mechanistic effects of 1D11 measured via μCT, biomechanical, dynamic histomorphometry, and serum biomarkers of bone turnover. In addition, osteoblast and osteoclast numbers in histological bone sections were assessed to better understand the mechanism of action of the 1D11 antibody in OI. Here we show that 1D11 treatment resulted in both dose and frequency dependency, increases in trabecular bone volume fraction and ultimate force in lumbar bone, and ultimate force, bending strength, yield force, and yield strength in the femur (p ≤ 0.05). Suppression of serum biomarkers of osteoblast differentiation, osteocalcin, resorption, CTx‐1, and bone formation were observed after 1D11 treatment of OI mice. Immunohistochemical analysis showed dose and frequency dependent decreases in runt‐related transcription factor, and increase in alkaline phosphatase in lumbar bone sections. In addition, a significant decrease in TRACP and the number of osteoclasts to bone surface area was observed with 1D11 treatment. Our results show that inhibition of the TGF‐β pathway corrects the high‐turnover aspects of bone disease and improves biomechanical properties of OI mice. These results highlight the potential for a novel treatment for osteogenesis imperfecta. © 2021 Sanofi‐Genzyme. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A systematic review and meta-analysis on the efficacy of stem cell therapy on bone brittleness in mouse models of osteogenesis imperfecta

There is no cure for osteogenesis imperfecta (OI), and current treatments can only partially correct the bone phenotype. Stem cell therapy holds potential to improve bone quality and quantity in OI. Here, we conduct a systematic review and meta-analysis of published studies to investigate the efficacy of stem cell therapy to rescue bone brittleness in mouse models of OI. Identified studies included bone marrow, mesenchymal stem cells, and human fetal stem cells. Effect size of fracture incidence, maximum load, stiffness, cortical thickness, bone volume fraction, and raw engraftment rates were pooled in a random-effects meta-analysis. Cell type, cell number, injection route, mouse age, irradiation, anatomical bone, and follow up time were considered as moderators. It was not possible to investigate further parameters due to the lack of standards of investigation between the studies. Despite the use of oim mice in the majority of the investigations considered and the lack of sham mice as control, this study demonstrates the promising potential of stem cell therapy to reduce fractures in OI. Although their low engraftment, cell therapy in mouse models of OI had a beneficial effect on maximum load, but not on stiffness, cortical thickness and bone volume. These parameters all depend on bone geometry and do not inform on its material properties. Being bone fractures the primary symptom of OI, there is a critical need to measure the fracture toughness of OI bone treated with stem cells to assess the actual efficacy of the treatment to rescue OI bone brittleness.