Osteogenesis Imperfecta Mouse Research Articles

Collagen mutation causes changes of the microdamage morphology in bone of an OI mouse model

Previous studies have postulated that ultrastructural changes may alter the pattern and capacity of microdamage accumulation in bone. Using an osteogenesis imperfecta (OI) mouse model, this study was performed to investigate the correlation of collagen mutation with the microdamage morphology and the associated brittleness of bone. In this study, femurs from mild OI and wild type mice were fatigued under four-point bending to create microdamage in the specimens. Then, the microdamage morphology of these specimens was examined using the bulk-staining technique with basic fuchsin. Similar with the results of previous studies, it was observed that linear microcracks were formed more easily in compression, whereas diffuse damage was induced more readily in tension for both wild-type and mild-type mice. However, less diffuse damage was found in the tensile side of mild OI mouse femurs (collagen mutation) compared with those of wild type mice, showing that the microdamage morphology is correlated to the brittleness of bone. The results of this study provide direct evidence that supports the prediction made by the previous numerical simulation studies, suggesting that microdamage morphology in bone is significantly correlated with the integrity of the collagen phase.

Alendronate Treatment of the Brtl Osteogenesis Imperfecta Mouse Improves Femoral Geometry and Load Response Before Fracture but Decreases Predicted Material Properties and Has Detrimental Effects on Osteoblasts and Bone Formation

Long courses of bisphosphonates are widely administered to children with osteogenesis imperfecta (OI), although bisphosphonates do not block mutant collagen secretion and may affect bone matrix composition or structure. The Brtl mouse has a glycine substitution in col1a1 and is ideal for modeling the effects of bisphosphonate in classical OI. We treated Brtl and wildtype mice with alendronate (Aln; 0.219 mg/kg/wk, SC) for 6 or 12 wk and compared treated and untreated femora of both genotypes. Mutant and wildtype bone had similar responses to Aln treatment. Femoral areal BMD and cortical volumetric BMD increased significantly after 12 wk, but femoral length and growth curves were unaltered. Aln improved Brtl diaphyseal cortical thickness and trabecular number after 6 wk and cross-sectional shape after 12 wk. Mechanically, Aln significantly increased stiffness in wildtype femora and load to fracture in both genotypes after 12 wk. However, predicted material strength and elastic modulus were negatively impacted by 12 wk of Aln in both genotypes, and metaphyseal remnants of mineralized cartilage also increased. Brtl femoral brittleness was unimproved. Brtl osteoclast and osteoblast surface were unchanged by treatment. However, decreased mineral apposition rate and bone formation rate/bone surface and the flattened morphology of Brtl osteoblasts suggested that Aln impaired osteoblast function and matrix synthesis. We conclude that Aln treatment improves Brtl femoral geometry and load to fracture but decreases bone matrix synthesis and predicted material modulus and strength, with striking retention of mineralized cartilage. Beneficial and detrimental changes appear concomitantly. Limiting cumulative bisphosphonate exposure of OI bone will minimize detrimental effects.

Quantitative Second Harmonic Generation Imaging of the Diseased State Osteogenesis Imperfecta: Experiment and Simulation

We report the integrated use of 3D second harmonic generation (SHG) imaging microscopy and Monte Carlo simulation as a combined metric to quantifiably differentiate normal and diseased tissues based on the physical properties of the respective extracellular matrix. To achieve this, we have identified a set of parameters comprised of the SHG creation attributes and the bulk optical parameters, which are used collectively via comparative analysis. Monte Carlo simulations of the SHG axial directional and attenuation responses allow their decomposition into the underlying factors that are not readily obtainable through experimental techniques. Specifically, this approach allows for estimation of the SHG creation attributes (directionality and relative conversion efficiency) and separation of primary and secondary filter effects, collectively that form the observed SHG contrast. The quantitative metric is shown for the connective tissue disorder Osteogenesis Imperfecta (characterized by abnormal assembly of type I collagen) using a murine model that expresses the disease in the dermis layer of skin. Structural dissimilarities between the osteogenesis imperfecta mouse and wild-type tissues lead to significant differences in the SHG depth-dependent directionality and signal attenuation. The Monte Carlo simulations of these responses using measured bulk optical parameters reproduce the experimental data trends, and the extracted emission directionality and conversion efficiencies are consistent with independent determinations. The simulations also illustrate the dominance of primary filter affects on overall SHG generation and attenuation. Thus, the combined method of 3D SHG imaging and modeling forms an essential foundation for parametric description of the matrix properties that are not distinguishable by sole consideration of either bulk optical parameters or SHG alone. Moreover, due to the quasi-coherence of the SHG process in tissues, we submit that this approach contains unique information not possible by purely scattering based methods and that these methods will be applicable in the general case where the complex fibrillar structure is difficult to fully quantify via morphological analysis.

Progenitors Systemically Transplanted into Neonatal Mice Localize to Areas of Active Bone Formation In Vivo: Implications of Cell Therapy for Skeletal Diseases

The potential of cell or gene therapy to treat skeletal diseases was evaluated through analysis of transplanted osteoprogenitors into neonatal homozygous and heterozygous osteogenesis imperfecta mice (oim). The osteoprogenitors used for transplantation were prepared by injection of mesenchymal stem cells (MSCs) marked with the green fluorescent protein (GFP) into normal mice with the subsequent retrieval of the cells at 35 days. The retrieved cells referred to here as osteoprogenitors were expanded in culture and transplanted into the 2-day-old oim mice via the superficial temporal vein. The recipient mice were evaluated at 2 and 4 weeks after cell transplantation. Four weeks after transplantation, tissue sections made from femurs and tibias of oim mice showed that the GFP-positive (GFP(+)) cells were distributed on the surfaces of the bone spicules in the spongiosa, the area of active bone formation. In the diaphysis, the GFP(+) cells were distributed in the bone marrow, on the endosteal surfaces, and also in the cortical bone. Immunofluorescence localization for GFP confirmed that the fluorescence seen in tissue sections was due to the engrafted donor cells, not bone autofluorescence. Gene expression analysis by polymerase chain reaction of the GFP(+) cells retrieved from the bones and marrow of the recipient mice demonstrated that the cells from bone were osteoblasts, whereas those from bone marrow were progenitors. These data demonstrate that MSCs delivered systemically to developing osteogenesis imperfecta mice engraft in bones, localize to areas of active bone formation, differentiate into osteoblasts in vivo, and may contribute to bone formation in vivo.

889. Lanthanum Enhances Adenovirus-Mediated Gene Transfer

Top of pageAbstract Introduction: In this study we investigated the use of lanthanum for improved efficiency of adenoviral gene transfer. Lanthanum ions, La 3+, are stable inorganic ions with a high charge to volume ratio enabling them to bind to negatively charged proteins with high efficiency. Methods: First generation adenoviruses encoding human Bone Morphogenetic Protein-2 (Ad-BMP-2) or Green Fluorescence Protein (Ad-GFP) cDNA were used. Osteoprogenitor cells were obtained from Osteogenesis Imperfecta Mice (OIM). La3+/virus complexes were prepared by adding the vectors to serum-free DMEM. LaCl3 was then added (200 |[mu]|M). Viral doses ( 10-300 MOI) were used for infection in 24-well plates. Non-lanthanum groups contained adenoviral vector and serum-free DMEM only. The viral mixtures were added to OIM cells and incubated. OIM cells in 24 well cell culture plates were infected with Ad-GFP with and without Lanthanum. (MOI = 300, 100, 30, 10). After 48 h, the samples were analyzed on FACScan. The induction of Alkaline Phosphatase Activity was assessed at 5 days. Results: Flow Cytometry showed, that Lanthanum enhanced transduction efficiency for all viral doses. The effect was highest at 10 MOI (12-fold increase) and diminished slightly with increasing viral dose (Fig. 1). The mean fluorescence intensity within transduced cells also increased in all lanthanum groups (Fig.2). However this effect decreased dramatically with viral dose, ranging from 18-fold at 300 MOI to only 3-fold at 10 MOI. To determine if La-Ad complexes could enhance a biological response, BMP-2 induction of Alkaline Phosphatase Activity was measured in OIM cells in the presence and absence of La3+. Alkaline phosphatase activity increased dose-dependently with Ad.BMP-2 infection, with a peak activity of 310 U/L at 100 MOI. Addition of La3+, increased alkaline phosphatase activity at 10 MOI and 30 MOI compared to virus only, suggesting enhanced adenoviral-mediated expression of BMP-2 at these doses. Moreover, equivalent levels of alkaline phosphatase activity, |[sim]|300 U/L, could be generated with 3-fold less viral vector in the presence of La3+. Discussion: Our findings indicate that the addition of lanthanum to adenoviral vectors can increase their transduction efficiency. This may allow for a reduction in the number of adenoviral particles, reducing viral production costs and immunological burden of virus.

In vivo expression of human growth hormone by genetically modified murine bone marrow stromal cells and its effect on the cells in vitro.

Human growth hormone (hGH) is frequently used clinically for growth abnormalities in children and also in adults with growth hormone deficiency. The hormone is usually administered to the individuals by frequent injections. In the present study we investigated the potential of bone marrow stromal cells as vehicles to deliver the GH in vivo by infusion of cells transduced with hGH cDNA into mice femurs. The effect of the hormone on the transduced cells in vitro was also assessed. Bone marrow stromal cells established from a mouse model of human osteogenesis imperfecta mice (oim) were transduced with a retrovirus containing hGH and neomycin resistance genes. The hGH-expressing cells were selected in a medium containing G418 and were then assessed for the hGH expression in vitro. The selected cells synthesized 15 ng/10(6) cells of hGH per 24 h in vitro and exhibited alkaline phosphatase activity when they were treated with the human recombinant bone morphogenetic protein 2 (rhBMP-2). The transduced cells also proliferated faster than the LacZ transduced cells but they did not exhibit a higher rate of matrix synthesis. When 2 x 10(6) hGH+ cells were injected into the femurs of mice, hGH was detected in the serum of the recipient mice up to 10 days after injection. The highest level of growth hormone expression, 750 pg/ml, was detected in the serum of the recipient mice I day after injection of the transduced cells. hGH was also detected in the medium conditioned by cells that were flushed from the femurs of the recipient mice at 1, 3, and 6 days after cell injection. These data indicate that bone marrow stromal cells could potentially be used therapeutically for the delivery of GH or any other therapeutic proteins targeted for bone. The data also suggest that GH may exert its effects on bone marrow stromal cells by increasing their rate of proliferation.

Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging.

Bone marrow stromal cells isolated from a model of osteogenesis imperfecta (oim) mice, were transduced with a retrovirus (BAG) carrying the LacZ and neor genes after passage 21. The transduced cells retained the ability to express alkaline phosphatase activity in vitro when treated with recombinant human bone morphogenetic protein two (rhBMP-2), formed cartilage in vitro in aggregate cultures and formed bone in ceramic cubes after 6 weeks of implantation in nude mice. X-gal staining of ceramic cubes seeded with the transduced cells demonstrated the presence of LacZ-positive cells on the edges of bone and also in the lacunae of the newly formed bone 6 weeks after implantation. After infusion into femurs of oim mice, the transduced cells were detected in the marrow cavity and on the edges of the trabecular bone of the injected and contralateral femurs by X-gal staining and PCR analysis at 4, 10, 20, 30 and 40 days after injection. The LacZ gene was also detected in the lung and liver of the recipient mice at 4 and 10 days after injection but not at later time-periods. The present findings suggest that long-term cultured bone marrow stromal cells from osteogenesis imperfecta (OI) animals have the potential to traffic through the circulatory system, home to bone, form bone and continue to express exogenous genes. These findings open the possibility of using these cells as vehicles to deliver normal genes to bone as an alternative approach for the treatment of some forms of OI and certain other bone acquired and genetic diseases.

Collagen from the osteogenesis imperfecta mouse model (oim) shows reduced resistance against tensile stress.

Osteogenesis imperfecta (OI) is a disease attributable to any of a large number of possible mutations of type I collagen. The disease is clinically characterized in part by highly brittle bone, the cause of this feature being unknown. Recently a mouse model of OI, designated as osteogenesis imperfecta murine (oim), and having a well defined genetic mutation, has been studied and found to contain mineral crystals different in their alignment with respect to collagen and in their size. These observations are consistent with those reported in human OI and the unusual crystal alignment and size undoubtedly contribute to the reduced mechanical properties of OI bone. While the mineral has been investigated, no information is available on the tensile properties of oim collagen. In this study, the mechanical properties of tendon collagen under tension have been examined for homozygous (oim/oim), heterozygous (+/oim), and control (+/+) mice under native wet conditions. The ultimate stress and strain found for oim/oim collagen were only about half the values for control mice. Assuming that prestrained collagen molecules carry most of the tensile load in normal bone while the mineral confers rigidity and compression stability, the reported results suggest that the brittleness of OI bone in the mouse model may be related to a dramatic reduction of the ultimate tensile strain of the collagen.

Bone mineralization in an osteogenesis imperfecta mouse model studied by small-angle x-ray scattering.

We have studied the size and orientation of mineral crystals in cortical bone of oim/oim mice, which are known to produce only alpha 1(I) collagen homotrimers and which may serve as a model for human osteogenesis imperfecta. Long bones (femur and tibia) from young (5 wk old) oim/oim mice and from unaffected heterozygous counterparts were investigated by small-angle x-ray scattering (SAXS), which is sensitive to structures smaller than 50 nm. Mineral crystals were compared in terms of their thickness and their alignment with respect to the long bone axis. While electron microscopic tomography has recently shown the existence of large mineral blocks (with all dimensions typically exceeding 50 nm) in mineralized tendons of oim/oim mice, SAXS revealed a family of thin, possibly needle-like, crystals in cortical bone. These crystals were similar in shape to those observed previously in normal mice, but they were thinner and less well aligned in oim/oim mice relative to heterozygotes. Moreover, the crystal thickness and their alignment with the bone axis were more variable in oim/oim bone, with a close correlation (r = 0.94, P < 0.001) between the two parameters. The presence of smaller crystals with more variable alignment in corticalis of oim/oim mice may contribute to the brittleness of their bone, similar to that of human osteogenesis imperfecta.

Mineral crystals are smaller and less oriented in osteogenesis imperfecta mouse model, as revealed by small-angle X-ray scattering

Mineral crystals are smaller and less oriented in osteogenesis imperfecta mouse model, as revealed by small-angle X-ray scattering

Methods for video image analysis of cancellous bone

Previous studies have postulated that ultrastructural changes may alter the pattern and capacity of microdamage accumulation in bone. Using an osteogenesis imperfecta (OI) mouse model, this study was performed to investigate the correlation of collagen mutation with the microdamage morphology and the associated brittleness of bone. In this study, femurs from mild OI and wild type mice were fatigued under four-point bending to create microdamage in the specimens. Then, the microdamage morphology of these specimens was examined using the bulk-staining technique with basic fuchsin. Similar with the results of previous studies, it was observed that linear microcracks were formed more easily in compression, whereas diffuse damage was induced more readily in tension for both wild-type and mild-type mice. However, less diffuse damage was found in the tensile side of mild OI mouse femurs (collagen mutation) compared with those of wild type mice, showing that the microdamage morphology is correlated to the brittleness of bone. The results of this study provide direct evidence that supports the prediction made by the previous numerical simulation studies, suggesting that microdamage morphology in bone is significantly correlated with the integrity of the collagen phase.