Improved in vivo osseointegration and degradation behavior of PEO surface-modified WE43 magnesium plates and screws after 6 and 12 months.

Magnesium is a highly promising candidate with respect to its future use as a material for resorbable implants. When magnesium degrades, hydrogen gas is released. High doses of gas emergence are reported to impair osseointegration and may therefore lead to fixation failure. The successful delay and reduction of the degradation rate by applying plasma electrolytic oxidation (PEO) as a post processing surface modification method for magnesium alloy has recently been demonstrated. The aim of this study was thus to compare the degradation behavior of a WE43-based plate system with and without respective PEO surface modification and to further investigate osseointegration, as well as the resulting effects on the surrounding bony tissue of both variants in a miniature pig model. WE43 magnesium screws and plates without (WE43) and with PEO surface modification (WE43-PEO) were implanted in long bones of Göttingen Miniature Pigs. At six and twelve months after surgery, micro-CT and histomorphometric analysis was performed. Residual screw volume (SV/TV; WE43: 28.8 ± 21.1%; WE43-PEO: 62.9 ± 31.0%; p = 0.027) and bone implant contact area (BIC; WE43: 18.1 ± 21.7%; WE43-PEO: 51.6 ± 27.7%; p = 0.015) were increased after six months among the PEO-modified implants. Also, surrounding bone density within the cortical bone was not affected by surface modification (BVTV; WE43: 76.7 ± 13.1%; WE43-PEO: 73.1 ± 16.2%; p = 0.732). Intramedullar (BV/TV; WE43: 33.2 ± 16.7%; WE43-PEO 18.4 ± 9.0%; p = 0.047) and subperiosteal (bone area; WE43: 2.6 ± 3.4 mm2; WE43-PEO: 6,9 ± 5.2 mm2; p = 0.049) new bone formation was found for both, surface-modified and non-surface-modified groups. After twelve months, no significant differences of SV/TV and BV/TV were found between the two groups. PEO surface modification of WE43 plate systems improved osseointegration and significantly reduced the degradation rate within the first six months in vivo. Osteoconductive and osteogenic stimulation by WE43 magnesium implants led to overall increased bone growth, when prior PEO surface modification was conducted.

Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin.

BACKGROUND: Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair. METHODS: Cortical injury was induced bilaterally in the tuberositas tibiae in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and in situ hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice. RESULTS: Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells. CONCLUSION: Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models.

Neurofibromin has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific inactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Here, we show that inactivation of the Nf1 gene also impairs osteocyte development. We analyzed cortical bone tissue in two conditional mouse models, Nf1Prx1 and Nf1Col1, for morphological and molecular effects. Backscattered electron microscopy revealed significantly enlarged osteocyte lacunae in Nf1Prx1 and Nf1Col1 mice (level E2: ctrl=1.90±0.52%, Nf1Prx1=3.40±0.95%; ctrl 1.60±0.47%, Nf1Col1 2.46±0.91%). Moreover, the osteocyte lacunae appeared misshaped in Nf1Prx1 and Nf1Col1 mice as indicated by increased Feret ratios. Strongest osteocyte and dendritic network disorganization was observed in proximity of muscle attachment sites in Nf1Prx1 humeri. In contrast to control cells, Nf1Prx1 osteocytes contained abundant cytosolic vacuoles and accumulated immature organic matrix within the perilacunar space, a phenotype reminiscent of the hyperosteoidosis shown Nf1 deficient mice. Cortical bone lysates further revealed approx. twofold upregulated MAPK signalling in osteocytes of Nf1Prx1 mice. This was associated with transcriptional downregulation of collagens and genes involved in mechanical sensing in Nf1Prx1 and Nf1Col1 bone tissue. In contrast, matrix gla protein (MGP), phosphate regulating endopeptidase homolog, X-linked (PHEX), and genes involved in lipid metabolism were upregulated. In line with previously described hyperactivation of Nf1 deficient osteoblasts, systemic plasma levels of the bone formation markers osteocalcin (OCN) and procollagen typ I N-propeptide (PINP) were approx. twofold increased in Nf1Prx1 mice. Histochemical and molecular analysis ascertained that osteocytes in Nf1Prx1 cortical bone were viable and did not undergo apoptosis or autophagy. We conclude that loss of neurofibromin is not only critical for osteoblasts but also hinders normal osteocyte development. These findings expand the effect of neurofibromin onto yet another cell type where it is likely involved in the regulation of mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.

Multiscale, converging defects of macro-porosity, microstructure and matrix mineralization impact long bone fragility in NF1.

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.

Autosomal recessive mental retardation, deafness, ankylosis, and mild hypophosphatemia associated with a novel ANKH mutation in a consanguineous family.

CONTEXT: Mutations in ANKH cause the highly divergent conditions familial chondrocalcinosis and craniometaphyseal dysplasia. The gene product ANK is supposed to regulate tissue mineralization by transporting pyrophosphate to the extracellular space. OBJECTIVE: We evaluated several family members of a large consanguineous family with mental retardation, deafness, and ankylosis. We compared their skeletal, metabolic, and serological parameters to that of the autosomal recessive progressive ankylosis (ank) mouse mutant, caused by a loss-of-function mutation in the murine ortholog Ank. PARTICIPANTS: The studied patients had painful small joint soft-tissue calcifications, progressive spondylarthropathy, osteopenia, mild hypophosphatemia, mixed hearing loss, and mental retardation. RESULTS: After mapping the disease gene to 5p15, we identified the novel homozygous ANK missense mutation L244S in all patients. Although L244 is a highly conserved amino acid, the mutated ANK protein was detected at normal levels at the plasma membrane in primary patient fibroblasts. The phenotype was highly congruent with the autosomal recessive progressive ankylosis (ank) mouse mutant. This indicates a loss-of-function effect of the L244S mutation despite normal ANK protein expression. Interestingly, our analyses revealed that the primary step of joint degeneration is fibrosis and mineralization of articular soft tissues. Moreover, heterozygous carriers of the L244S mutation showed mild osteoarthritis without metabolic alterations, pathological calcifications, or central nervous system involvement. CONCLUSION: Beyond the description of the first human progressive ankylosis phenotype, our results indicate that ANK influences articular soft tissues commonly involved in degenerative joint disorders. Furthermore, this human disorder provides the first direct evidence for a role of ANK in the central nervous system.