Craniofacial dysmorphism of osteogenesis imperfecta mouse and effect of cathepsin K knockout: Preliminary craniometry observations.

OBJECTIVES: In addition to bone fragility, patients with osteogenesis imperfecta (OI) type III have typical craniofacial abnormalities, such as a triangular face and maxillary micrognathism. However, in the osteogenesis imperfecta mouse (oim), a validated model of OI type III, few descriptions exist of craniofacial phenotype. Treatment of OI mostly consists of bisphosphonate administration. Cathepsin K inhibition has been tested as a promising therapeutic approach for osteoporosis and positive results were observed in long bones of cathepsin K knocked out oim (oim/CatK-/-). This craniometry study aimed to highlight the craniofacial characteristics of oim and Cathepsin K KO mouse. MATERIALS AND METHODS: We analyzed the craniofacial skeleton of 51 mice distributed in 4 genotype groups: Wt (control), oim, CatK-/-, oim/CatK-/-. The mice were euthanized at 13 weeks and their heads were analyzed using densitometric (pQCT), X-ray cephalometric, and histomorphometric methods. RESULTS: The craniofacial skeleton of the oim mouse is frailer than the Wt one, with a reduced thickness and mineral density of the cranial vault and mandibular ramus. Different cephalometric data attest a dysmorphism similar to the one observed in humans with OI type III. Those abnormalities were not improved in the oim/CatK-/- group. CONCLUSION: These results suggest that oim mouse could serve as a complete model of the human OI type III, including the craniofacial skeleton. They also suggest that invalidation of cathepsin K has no impact on the craniofacial abnormalities of the oim model.

Dental tissue changes in juvenile and adult mice with osteogenesis imperfecta.

Osteogenesis imperfecta (OI), a disorder of type I collagen, causes skeletal deformities as well as defects in dental tissues, which lead to increased enamel wear and smaller teeth with shorter roots. Mice with OI exhibit similar microstructural dentin changes, including reduced dentin tubule density and dentin cross-sectional area. However, the effects of these mutations on gross dental morphology and dental tissue volumes have never been characterized in the osteogenesis imperfecta murine (OIM) mouse model. Here we compare mineralized dental tissue measurements of OIM mice and unaffected wild type (WT) littermates at the juvenile and adult stages. The maxillary and mandibular incisors and first molars were isolated from microCT scans, and tissue volumes and root length were measured. OIM mice have smaller teeth with shorter roots relative to WT controls. Maxillary incisor volumes differed significantly between OIM and WT mice at both the juvenile and young adult stage, perhaps due to shortening of the maxilla itself in OIM mice. Additionally, adult OIM mice have significantly less crown enamel volume than do juveniles, potentially due to loss through wear. Thus, OIM mice demonstrate a dental phenotype similar to humans with OI, with decreased tooth size, decreased root length, and accelerated enamel wear. Further investigation of dental development in the OIM mouse may have important implications for the development and treatment of dental issues in OI patients.

Col1A-2 Mutation in Osteogenesis Imperfecta Mice Contributes to Long Bone Fragility by Modifying Cell-Matrix Organization.

Osteogenesis imperfecta (OI) is a rare congenital bone dysplasia generally caused by a mutation of one of the type I collagen genes and characterized by low bone mass, numerous fractures, and bone deformities. The collagen organization and osteocyte lacuna arrangement were investigated in the long bones of 17-week-old wildtype (WT, n = 17) and osteogenesis imperfecta mice (OIM, n = 16) that is a validated model of severe human OI in order to assess their possible role in bone fragility. Fractures were counted after in vivo scanning at weeks 5, 11, and 17. Humerus, femur, and tibia diaphyses from both groups were analyzed ex vivo with pQCT, polarized and ordinary light histology, and Nano-CT. The fractures observed in the OIM were more numerous in the humerus and femur than in the tibia, whereas the quantitative bone parameters were altered in different ways among these bones. Collagen fiber organization appeared disrupted, with a lower birefringence in OIM than WT bones, whereas the osteocyte lacunae were more numerous, more spherical, and not aligned in a lamellar pattern. These modifications, which are typical of immature and less mechanically competent bone, attest to the reciprocal alteration of collagen matrix and osteocyte lacuna organization in the OIM, thereby contributing to bone fragility.

Beneficial Effects of Zoledronic Acid on Tendons of the Osteogenesis Imperfecta Mouse (Oim).

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by spontaneous fractures, bone deformities, impaired growth and posture, as well as extra-skeletal manifestations. Recent studies have underlined an impairment of the osteotendinous complex in mice models of OI. The first objective of the present work was to further investigate the properties of tendons in the osteogenesis imperfecta mouse (oim), a model characterized by a mutation in the COL1A2 gene. The second objective was to identify the possible beneficial effects of zoledronic acid on tendons. Oim received a single intravenous injection of zoledronic acid (ZA group) at 5 weeks and were euthanized at 14 weeks. Their tendons were compared with those of untreated oim (oim group) and control mice (WT group) by histology, mechanical tests, western blotting and Raman spectroscopy. The ulnar epiphysis had a significantly lower relative bone surface (BV/TV) in oim than WT mice. The tendon of the triceps brachii was also significantly less birefringent and displayed numerous chondrocytes aligned along the fibers. ZA mice showed an increase in BV/TV of the ulnar epiphysis and in tendon birefringence. The tendon of the flexor digitorum longus was significantly less viscous in oim than WT mice; in ZA-treated mice, there was an improvement of viscoelastic properties, especially in the toe region of stress-strain curve, which corresponds to collagen crimp. The tendons of both oim and ZA groups did not show any significant change in the expression of decorin or tenomodulin. Finally, Raman spectroscopy highlighted differences in material properties between ZA and WT tendons. There was also a significant increase in the rate of hydroxyproline in the tendons of ZA mice compared with oim ones. This study highlighted changes in matrix organization and an alteration of mechanical properties in oim tendons; zoledronic acid treatment had beneficial effects on these parameters. In the future, it will be interesting to better understand the underlying mechanisms which are possibly linked to a greater solicitation of the musculoskeletal system.

Dysfunction of Caveolae-Mediated Endocytic TßRI Degradation Results in Hypersensitivity of TGF-ß/Smad Signaling in Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder caused by mutations of type I collagen-related genes, and excessive transforming growth factor-beta (TGF-ß) signaling is a common mechanism. TGF-ß/Smad signaling has inhibitory effects on osteoblast differentiation and maturation and is mainly transduced and regulated by the internalization of a tetrameric receptor complex comprising types I and II TGF-ß receptors (TßRI and TßRII). During internalization, clathrin-mediated endocytosis enhances TGF-ß/Smad signaling via Smad2/3 phosphorylation and receptors recycling, while caveolae-mediated endocytosis turns off TGF-ß/Smad signaling by promoting receptor ubiquitination and degradation. In this study, using an animal model of OI (Colla2oim , osteogenesis imperfecta murine [oim]/oim mouse), we found that osteoblastic cells of oim/oim mice were more sensitive to the inhibitory effects of TGF-ß on osteoblast differentiation and maturation and had much higher cell membrane protein levels of TGF-ß receptors than those of wild-type (wt)/wt mice. Further results showed that clathrin-mediated endocytosis of TßRI was enhanced, whereas caveolae-mediated TßRI endocytic degradation was reduced in oim/oim mice, combined with reduced caveolin-1 (Cav-1) phosphorylation. In addition, type I collagen downregulated TßRI via focal adhesion kinase (FAK) and Src activation-dependent Cav-1 phosphorylation. To further examine this mechanism, 4-week-old oim/oim and wt/wt mice were treated with either TßRI kinase inhibitor (SD-208) or vehicle for 8 weeks. SD-208 treatment significantly reduced the fracture incidence in oim/oim mice. Micro-computed tomography and biomechanical testing showed that femoral bone mass and strength were significantly improved with SD-208 treatment in both genotypes. Additionally, SD-208 significantly promoted osteoblast differentiation and bone formation and inhibited bone resorption. In conclusion, dysfunction of caveolae-mediated endocytic TßRI degradation is a possible mechanism for the enhanced TGF-ß/Smad signaling in OI. Targeting this mechanism using a TßRI kinase inhibitor effectively reduced fractures and improved bone mass and strength in OI model and, thus, may offer a new strategy for the treatment of OI. © 2022 American Society for Bone and Mineral Research (ASBMR).

Biomechanical, Microstructural and Material Properties of Tendon and Bone in the Young Oim Mice Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by low bone mass and spontaneous fractures, as well as extra-skeletal manifestations, such as dental abnormalities, blue sclera, hearing loss and joint hypermobility. Tendon ruptures have been reported in OI patients. Here, we characterized the biomechanical, structural and tissue material properties of bone and tendon in 5-week-old female osteogenesis imperfecta mice (oim), a validated model of severe type III OI, and compared these data with age- and sex-matched WT littermates. Oim tendons were less rigid and less resistant than those of WT mice. They also presented a significantly higher rate of pentosidine, without significant modification of enzymatic crosslinking. The oim bones were less resistant and avulsion fractures were evident at high tendinous stress areas. Alterations of trabecular and cortical bone microarchitectures were noticed in young female oim. Bone tissue material properties were also modified, with a less mature and more mineralized matrix in association with lower collagen maturity. Our data suggest that the tendon-to-bone unit is affected in young oim mice, which could explain tendon ruptures and bone fragility observed in OI patients.

Comparison of microscopic techniques to study the diversity of the bitumen microstructure.

Bitumen characterisation and differentiation usually involve a combination of mechanical and chemical analyses. However, these methods provide limited information on the diversity caused by the binders' origin or processing method. Thus, the question arises whether the bitumen microstructure can be used to identify these issues. In this study, microscopic methods, including brightfield, darkfield and fluorescence optical inverse microscopy (OIM), as well as confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM), were used to investigate the bitumen surface. Five different binders varying in their origin and production method were selected. The results show that CLSM, AFM and OIM darkfield can adequately capture a specific surface microstructure known as the bee structure, whereas brightfield in the OIM and optical CLSM show the surrounding peri phase, which exhibits a strong fluorescence. All visbroken binders show bee structures surrounded by a pronounced peri phase. On the other hand, one of the straight distilled binders does not show any microstructure, while the second straight distilled binder displays smaller bee structures surrounded by a small peri phase. Results from the image processing evaluation reveal that the area covered by bee structures is in the range of 2.4 - 4.3% for those binders that developed a surface microstructure. These results indicate a good accordance between the three microscopic techniques selected. However, a clear differentiation between the binders is difficult to obtain. Nonetheless, this work shows how these techniques can be used to their maximum capabilities regarding the obtained microstructural information and may help solve future questions regarding ageing, modification or rejuvenation.

Impact of the change of the Atalah standard cut-off point to classify underweight nutritional status during pregnancy

ABSTRACT Chile, and several Latin American countries, use the Atalah standard to assess nutritional status during pregnancy. However, this standard (underweight: pre-pregnancy body mass index (BMI)<20 kg/m2 and normal weight: pre-pregnancy BMI= 20-24.9 kg/m2) differ from those recommended by the US Institute of Medicine (IOM2009) (underweight: BMI<18.5 kg/m2 and normal weight: 18.5-24.9 kg/m2). Using a large population database from a Chilean public hospital, we compared the prevalence of underweight and normal weight at the beginning of pregnancy with Atalah and IOM2009 standards. Additionally, we evaluated the performance of both standards in detecting adverse neonatal outcomes and gestational weight gain. Methods: Data from clinical records of single birth pregnancies (n= 59,476) at the Sótero del Río Hospital, between 2003-2012 were collected. We compared 1. nutritional status, 2. proportion of excessive gestational weight gain, 3. association between nutritional status and neonatal outcomes (large/small for gestational age, low birth weight, preterm birth and macrosomia), using logistic regression models, and 4. Sensitivity, specificity, and predictive values to predict adverse neonatal outcomes per nutritional status. Results: Pre-pregnancy underweight decreased from 8.6% to 2.5% and women with BMI between 18.5-19.9kg/m2, who exceeded the recommended gestational weight gain increased from 32.7% to 49.2% when using IOM2009 instead of Atalah. Both standards showed low sensitivity, but the IOM2009 cut-off points showed better specificity for identifying healthy newborns. Conclusion: The cut-off points recommended by the IOM2009 better identify the prevalence of underweight and normal weight during pregnancy without increasing neonatal risk. This study supports the recent change of the Ministry of Health in adopting the WHO cut-off points during pregnancy.

RESUMEN Chile y diversos países Latinoaméricanos utilizan el estándar de Atalah para evaluar el estado nutricional (EN) durante el embarazo. Sin embargo, los puntos de corte de este estándar (bajo peso, BP: índice de masa corporal pre-gestacional (IMC)<20 kg/m2 y normal, NP: IMC pregestacional= 20-24,9 kg/m2) difieren de los recomendados por el Instituto de Medicina de EE.UU. (IOM2009) (BP: BMI<18.5kg/m2 and NP: 18,5-24,9 kg/m2). Con datos obtenidos desde el Hospital Sótero del Río, nosotros evaluamos la prevalencia de BP y NP al comienzo del embarazo con los estándares de Atalah e IOM2009. Adicionalmente, nosotros comparamos el comportamiento de ambos estándares en detectar resultados neonatales (RN) adversos y en la clasificación de la ganancia de peso gestacional (GPG). Métodos: Se obtuvieron datos de embarazos simples entre 2003-2012, (n= 59.476). Nosotros comparamos: 1. Prevalencia de EN, 2. Proporción de excesiva GPG, 3. Asociación entre EN y RN, usando modelos de regresión logística, y 4. Sensibilidad, especificidad y valores predictivos para predecir RN según EN. Resultados: La prevalencia de BP pregestacional disminuyó de 8,6% a 2,5% y las mujeres con IMC entre 18,5-19,9 kg/m2 que excedieron la GPG recomendada, incrementaron desde 33% a 50% cuando se utilizó el estándar IOM2009 en vez de Atalah. Ambos estándares mostraron baja sensibilidad, pero IOM2009 mostró mejor especificidad para identificar recién nacidos saludables. Conclusión: Estandar IOM2009 identifica con mayor precisión la prevalencia de BP y NP durante la gestación sin incrementar el riesgo neonatal. Este estudio respalda el reciente cambio del MINSAL al adoptar los puntos de corte de la OMS durante el embarazo.

Gender-Related Impact of Sclerostin Antibody on Bone in the Osteogenesis Imperfecta Mouse.

Osteogenesis imperfecta (OI), which is most often due to a collagen type 1 gene mutation, is characterized by low bone density and bone fragility. In OI patients, gender-related differences were reported, but data in the literature are not convergent. We previously observed that sclerostin antibody (Scl-Ab), which stimulates osteoblast Wnt pathway via sclerostin inactivation, improved spine and long-bone parameters and biomechanical strength in female oim/oim mice, a validated model of human type 3 OI. Here, we wanted to highlight the effect of Scl-Ab on male oim/oim bones in order to identify a possible distinct therapeutic effect from that observed in females. According to the same protocol as our previous study with female mice, male wild-type (Wt) and oim/oim mice received vehicle or Scl-Ab from 5 to 14 weeks of age. Clinimetric and quantitative bone parameters were studied using X-rays, peripheral quantitative computed tomography, microradiography, and dynamic histomorphometry and compared to those of females. Contrary to Wt mice, male oim/oim had significantly lower weight, snout-sacrum length, and bone mineral content than females at 5 weeks. No significant difference in these clinimetric parameters was observed at 14 weeks, whereas male oim showed significantly more long-bone fractures than females. Scl-Ab improved bone mineral density and bone volume/total volume ratio (BV/TV) of vertebral body in Wt and oim/oim, without significant difference between male and female at 14 weeks. Male vehicle oim/oim had a significantly lower cortical thickness (Ct.Th) and BV/TV of tibial diaphysis than female and showed a higher number of fractures at 14 weeks. Scl-Ab increased midshaft periosteal apposition rate in such a way that tibial Ct.Th of male oim/oim was not significantly different from the female one at 14 weeks. The number of fractures was lower in male than female oim/oim after 14 weeks of Scl-Ab treatment, but this difference was not significant. Nevertheless, Scl-Ab-treated oim/oim male and female mice remained smaller than the Wt ones. In conclusion, our results highlighted differences between male and female oim/oim at 4 and 14 weeks of age, as well as some male-specific response of cortical bone to Scl-Ab. These gender-related particularities of oim/oim should be considered when testing experimental treatments.

Increased cochlear otic capsule thickness and intracortical canal porosity in the oim mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI or brittle bone disease) is a group of genetic disorders of the connective tissues caused mainly by mutations in the genes encoding collagen type I. Clinical manifestations of OI include skeletal fragility, bone deformities, and severe functional disabilities, such as hearing loss. Progressive hearing loss, usually beginning in childhood, affects approximately 70% of people with OI with more than half of the cases involving the inner ear. There is no cure for OI nor a treatment to ameliorate its corresponding hearing loss, and very little is known about the properties of OI ears. In this study, we investigate the morphology of the otic capsule and the cochlea in the inner ear of the oim mouse model of OI. High-resolution 3D images of 8-week old oim and WT inner ears were acquired using synchrotron microtomography. Volumetric morphometric measurements were conducted for the otic capsule, its intracortical canal network and osteocyte lacunae, and for the cochlear spiral ducts. Our results show that the morphology of the cochlea is preserved in the oim ears at 8 weeks of age but the otic capsule has a greater cortical thickness and altered intracortical bone porosity, with a larger number and volume density of highly branched canals in the oim otic capsule. These results portray a state of compromised bone quality in the otic capsule of the oim mice that may contribute to their hearing loss.

Intrafibrillar mineralization deficiency and osteogenesis imperfecta mouse bone fragility.

Osteogenesis imperfecta (OI), a brittle bone disease, is known to result in severe bone fragility. However, its ultrastructural origins are still poorly understood. In this study, we hypothesized that deficient intrafibrillar mineralization is a key contributor to the OI induced bone brittleness. To test this hypothesis, we explored the mechanical and ultrastructural changes in OI bone using the osteogenesis imperfecta murine (oim) model. Synchrotron X-ray scattering experiments indicated that oim bone had much less intrafibrillar mineralization than wild type bone, thus verifying that the loss of mineral crystals indeed primarily occurred in the intrafibrillar space of oim bone. It was also found that the mineral crystals were organized from preferentially in longitudinal axis in wild type bone to more randomly in oim bone. Moreover, it revealed that the deformation of mineral crystals was more coordinated with collagen fibrils in wild type than in oim bone, suggesting that the load transfer deteriorated between the two phases in oim bone. The micropillar test revealed that the compression work to fracture of oim bone (8.2 ± 0.9 MJ/m3) was significantly smaller (p < 0.05) than that of wild type bone (13.9 ± 2.7 MJ/m3), while the bone strength was not statistically different (p > 0.05) between the two genotype groups. In contrast, the uniaxial tensile test showed that the ultimate strength of wild type bone (50 ± 4.5 MPa) was significantly greater (p < 0.05) than that of oim bone (38 ± 5.3 MPa). Furthermore, the nanoscratch test showed that the toughness of oim bone was much less than that of wild type bone (6.6 ± 2.2 GJ/m3 vs. 12.6 ± 1.4 GJ/m3). Finally, in silico simulations using a finite element model of sub-lamellar bone confirmed the links between the reduced intrafibrillar mineralization and the observed changes in the mechanical behavior of OI bone. Taken together, these results provide important mechanistic insights into the underlying cause of poor mechanical quality of OI bone, thus pave the way toward future treatments of this brittle bone disease.

Combined approach for intrapelvic abscess drainage among adults.

Obturator internus muscle (OIM) and adductor brevis (AB) abscess occurs rarely in adults. This condition mainly affects children experiencing trauma. This paper presents and discusses a unique case of OIM and AB abscess in an adult male that was treated operatively with a combined surgical approach (modified Stoppa and Smith-Peterson) after multiple failed drainage procedures. CASE: A 23-year-old male underwent reduction and fixation of his right tibiae shaft fracture with an intramedullary nail. After several referrals to the emergency room due to recurrent symptoms of fever and groin pain, he was finally admitted and diagnosed with OIM and AB abscess and fulminant MRSA sepsis. The operative treatment was performed by addressing the abscess with an unusual combined surgical approach (Smith-Peterson and modified Stoppa) for abscess drainage. METHODS: Using PubMed, Google scholar and Hadassah Hebrew University search engines, we conducted a literature search using the following keywords: 'pyomyositis', 'obturator abscess', 'pelvic abscess', and 'pelvic osteomyelitis among children and adults'. Twenty case studies were found (14 children and 6 adults). No cases of combined OIM and AB abscess were found and only one OIM case was reported with methicillin-resistant Staphylococcus (MRSA).

Sclerostin-Antibody Treatment Decreases Fracture Rates in Axial Skeleton and Improves the Skeletal Phenotype in Growing oim/oim Mice.

In osteogenesis imperfecta (OI), vertebrae brittleness causes thorax deformations and leads to cardiopulmonary failure. As sclerostin-neutralizing antibodies increase bone mass and strength in animal models of osteoporosis, their administration in two murine models of severe OI enhanced the strength of vertebrae in growing female Crtap-/- mice but not in growing male Col1a1Jrt/+ mice. However, these two studies ignored the impact of antibodies on spine growth, fracture rates, and compressive mechanical properties. Here, we conducted a randomized controlled trial in oim/oim mice, an established model of human severe OI type III due to a mutation in Col1a2. Five-week-old female WT and oim/oim mice received either PBS or sclerostin antibody (Scl-Ab) for 9 weeks. Analyses included radiography, histomorphometry, pQCT, microcomputed tomography, and biomechanical testing. Though it did not modify vertebral axial growth, Scl-Ab treatment markedly reduced the fracture prevalence in the pelvis and caudal vertebrae, enhanced osteoblast activity (L4), increased cervico-sacral spine BMD, and improved the lumbosacral spine bone cross-sectional area. Scl-Ab did not impact vertebral height and body size but enhanced the cortical thickness and trabecular bone volume significantly in the two Scl-Ab groups. At lumbar vertebrae and tibial metaphysis, the absolute increase in cortical and trabecular bone mass was higher in Scl-Ab WT than in Scl-Ab oim/oim. The effects on trabecular bone mass were mainly due to changes in trabecular number at vertebrae and in trabecular thickness at metaphyses. Additionally, Scl-Ab did not restore a standard trabecular network, but improved bone compressive ultimate load with more robust effects at vertebrae than at metaphysis. Overall, Scl-Ab treatment may be beneficial for reducing vertebral fractures and spine deformities in patients with severe OI.

Sclerostin antibody reduces long bone fractures in the oim/oim model of osteogenesis imperfecta.

Osteogenesis imperfecta type III (OI) is a serious genetic condition with poor bone quality and a high fracture rate in children. In a previous study, it was shown that a monoclonal antibody neutralizing sclerostin (Scl-Ab) increases strength and vertebral bone mass while reducing the number of axial fractures in oim/oim, a mouse model of OI type III. Here, we analyze the impact of Scl-Ab on long bones in OI mice. After 9 weeks of treatment, Scl-Ab significantly reduced long bone fractures (3.6 ±â€¯0.3 versus 2.1 ±â€¯0.8 per mouse, p < 0.001). In addition, the cortical thickness of the tibial midshaft was increased (+42%, p < 0.001), as well as BMD (+28%, p < 0.001), ultimate load (+86%, p < 0.05), plastic energy (+184%; p < 0.05) and stiffness (+172%; p < 0.01) in OI Scl-Ab mice compared to OI vehicle controls. Similar effects of Scl-Ab were observed in Wild type (Wt) mice. The plastic energy, which reflects the fragility of the tissue, was lower in the OI than in the Wt and significantly improved with the Scl-Ab treatment. At the tissue level by nanoindentation, Scl-Ab slightly increased the elastic modulus in bones of both OI and Wt, while moderately increasing tissue hardness (+13% compared to the vehicle; p < 0.05) in Wt bones, but not in OI bones. Although it did not change the properties of the OI bone matrix material, Scl-Ab reduced the fracture rate of the long bones by improving its bone mass, density, geometry, and biomechanical strength. These results suggest that Scl-Ab can reduce long-bone fractures in patients with OI.

Studies of chain substitution caused sub-fibril level differences in stiffness and ultrastructure of wildtype and oim/oim collagen fibers using multifrequency-AFM and molecular modeling.

Molecular alteration in type I collagen, i.e., substituting the α2 chain with α1 chain in tropocollagen molecule, can cause osteogenesis imperfecta (OI), a brittle bone disease, which can be represented by a mouse model (oim/oim). In this work, we use dual-frequency Atomic Force Microscopy (AFM) and incorporated with molecular modeling to quantify the ultrastructure and stiffness of the individual native collagen fibers from wildtype (+/+) and oim/oim diseased mice humeri. Our work presents direct experimental evidences that the +/+ fibers have highly organized and compact ultrastructure and corresponding ordered stiffness distribution. In contrast, oim/oim fibers have ordered but loosely packed ultrastructure with uncorrelated stiffness distribution, as well as local defects. The molecular model also demonstrates the structural and molecular packing differences between +/+ and oim/oim collagens. The molecular mutation significantly altered sub-fibril structure and mechanical property of collagen fibers. This study can give the new insight for the mechanisms and treatment of the brittle bone disease.

Strontium Ranelate Reduces the Fracture Incidence in a Growing Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by brittle bones with increased fracture risk. Although current treatment options to improve bone strength in OI focus on antiresorptive bisphosphonates, controlled clinical trials suggest they have an equivocal effect on reducing fracture risk. Strontium ranelate (SrR) is a promising therapy with a dual mode of action that is capable of simultaneously maintaining bone formation and reducing bone resorption, and may be beneficial for the treatment of OI. In this study, SrR therapy was investigated to assess its effects on fracture frequency and bone mass and strength in an animal model of OI, the oim/oim mouse. Three-week-old oim/oim and wt/wt mice were treated with either SrR or vehicle (Veh) for 11 weeks. After treatment, the average number of fractures sustained by SrR-treated oim/oim mice was significantly reduced compared to Veh-treated oim/oim mice. Micro-computed tomographic (µCT) analyses of femurs showed that both trabecular and cortical bone mass were significantly improved with SrR treatment in both genotypes. SrR significantly inhibited bone resorption, whereas bone formation indices were maintained. Biomechanical testing revealed improved bone structural properties in both oim/oim and wild-type (wt/wt) mice under the treatment, whereas no significant effects on bone brittleness and material quality were observed. In conclusion, SrR was able to effectively reduce fractures in oim/oim mice by improving bone mass and strength and thus represents a potential therapy for the treatment of pediatric OI. © 2015 American Society for Bone and Mineral Research.

Raloxifene reduces skeletal fractures in an animal model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disease of Type I collagen and collagen-associated pathways that results in brittle bone behavior characterized by fracture and reduced mechanical properties. Based on previous work in our laboratory showing that raloxifene (RAL) can significantly improve bone mechanical properties through non-cellular mechanisms, we hypothesized that raloxifene would improve the mechanical properties of OI bone. In experiment 1, tibiae from female wild type (WT) and homozygous oim mice were subjected to in vitro soaking in RAL followed by mechanical tests. RAL soaking resulted in significantly higher post-yield displacement (+75% in WT, +472% in oim; p<0.004), with no effect on ultimate load or stiffness, in both WT and oim animals. In experiment 2, eight-week old WT and oim male mice were treated for eight weeks with saline vehicle (VEH) or RAL. Endpoint measures included assessment of in vivo skeletal fractures, bone density/geometry and mechanical properties. In vivo skeletal fractures of the femora, assessed by micro CT imaging, were significantly lower in oim-RAL (20%) compared to oim-VEH (48%, p=0.047). RAL led to significantly higher DXA-based BMD (p<0.01) and CT-based trabecular BV/TV in both WT and oim animals compared to those treated with VEH. Fracture toughness of the femora was lower in oim mice compared to WT and improved with RAL in both genotypes. These results suggest that raloxifene reduces the incidence of fracture in this mouse model of oim. Furthermore, they suggest that raloxifene's effects may be the result of both cellular (increased bone mass) and non-cellular (presumably changes in hydration) mechanisms, raising the possibility of using raloxifene, or related compounds, as a new approach for treating bone fragility associated with OI.

Altered lacunar and vascular porosity in osteogenesis imperfecta mouse bone as revealed by synchrotron tomography contributes to bone fragility.

Osteogenesis imperfecta (brittle bone disease) is caused by mutations in the collagen genes and results in skeletal fragility. Changes in bone porosity at the tissue level indicate changes in bone metabolism and alter bone mechanical integrity. We investigated the cortical bone tissue porosity of a mouse model of the disease, oim, in comparison to a wild type (WT-C57BL/6), and examined the influence of canal architecture on bone mechanical performance. High-resolution 3D representations of the posterior tibial and the lateral humeral mid-diaphysis of the bones were acquired for both mouse groups using synchrotron radiation-based computed tomography at a nominal resolution of 700nm. Volumetric morphometric indices were determined for cortical bone, canal network and osteocyte lacunae. The influence of canal porosity architecture on bone mechanics was investigated using microarchitectural finite element (µFE) models of the cortical bone. Bright-field microscopy of stained sections was used to determine if canals were vascular. Although total cortical porosity was comparable between oim and WT bone, oim bone had more numerous and more branched canals (p<0.001), and more osteocyte lacunae per unit volume compared to WT (p<0.001). Lacunae in oim were more spherical in shape compared to the ellipsoidal WT lacunae (p<0.001). Histology revealed blood vessels in all WT and oim canals. µFE models of cortical bone revealed that small and branched canals, typical of oim bone, increase the risk of bone failure. These results portray a state of compromised bone quality in oim bone at the tissue level, which contributes to its deficient mechanical properties.

Epigenetic regulation of osteogenesis: human embryonic palatal mesenchymal cells.

Mesenchymal stem cells (MSCs) provide an appropriate model to study epigenetic changes during osteogenesis and bone regeneration due to their differentiation potential. Since there are no unique markers for MSCs, methods of identification are limited. The complex morphology of human embryonic palatal mesenchyme stem cell (HEPM) requires analysis of fractal dimensions to provide an objective quantification of self-similarity, a statistical transformation of cellular shape and border complexity. We propose the hypothesis of a study to compare and contrast sequential steps of osteogenic differentiation in HEPMs both phenotypically using immunocytochemistry, and morphometrically using fractal analysis from undifferentiated passage 1 (P1) to passage 7 (P7) cells. The proof-of-concept is provided by results we present here that identify and compare the modulation of expression of certain epigenetic biomarkers (alkaline phosphatase, ALP; stromal interaction molecule-1, STRO-1; runt-related transcription factor-2, RUNX2), which are established markers of osteogenesis in bone marrow studies, of osteoblastic/skeletal morphogenesis, and of osteoblast maturation. We show that Osteoinductive medium (OIM) modulates the rate of differentiation of HEPM into Run-2+ cells, the most differentiated subpopulation, followed by ALP+ and STRO-1+ cells. Taken together, our phenotypical and morphometric data demonstrate the feasibility of using HEPM to assess osteogenic differentiation from an early undifferentiated to a differentiated stage. This research model may lay the foundation for future studies aimed at characterizing the epigenetic characteristics of osteoimmunological disorders and dysfunctions (e.g., osteoarthritis, temporomandibular joint disorders), so that proteomic profiling can aid the diagnosis and monitor the prognosis of these and other osteoimmunopathologies.

RANKL inhibition improves bone properties in a mouse model of osteogenesis imperfecta.

Recently, a new class of agents targeting the receptor activator of nuclear factor-kappaB ligand (RANKL) pathway has been developed for the treatment of osteoporosis and other bone diseases. In the current study, inhibition of the RANKL pathway was evaluated to assess effects on "bone quality" and fracture incidence in an animal model of osteogenesis imperfect (OI), the oim/oim mouse. Juvenile oim/oim ( approximately 6 weeks old) and wildtype (+/+) mice were treated with either a RANKL inhibitor (RANK-Fc) or saline. After treatment, bone density increased significantly in the femurs of both genotypes. Femoral length decreased with RANK-Fc in +/+ mice. Geometric measurements at mid-diaphysis in the oim/oim groups showed increases in the ML periosteal and endosteal diameters and AP cortical thickness in the treated groups. Within +/+ groups, ML cortical thickness and ML femoral periosteal diameter were significantly increased with RANK-Fc. Biomechanical testing revealed increased stiffness in oim/oim and +/+ mice. Total strain was increased with treatment in the +/+ mice. Histologically, RANKL inhibition resulted in retained growth plate cartilage in both genotypes. The average number of fractures sustained by RANK-Fc-treated oim/oim mice was not significantly decreased compared to saline treated oim/oim mice. This preclinical study demonstrated that RANKL inhibition at the current dose improved density and some geometric and biomechanical properties of oim/oim bone, but it did not decrease fracture incidence. Further studies that address commencement of therapy at earlier time points are needed to determine whether this mode of therapy will be clinically useful in OI.