Figure 1.
Nf1Prx1 mice show increased macro-porosity and ectopic blood vessels associated mineralization defects.
(A–C) High-resolution micro-CT (skyscan) and von Kossa/Masson-Goldner histology of adult humeri. Consecutive cross-sections of the mid-shaft region (E2) in Nf1Prx1, Nf1Col1 and control mice representing following morphological sites: deltoid tuberosity (1), nutrient artery (2), bone cortex between deltoid tuberosity and epicondylus medialis (3), left cortex (4), and right cortex (5). (A) (A1) Deltoid tuberosity of controls showed solid appearance and smooth mineralization front. (A2) The site of nutrient artery bone cortex traverse. Note a fully mineralized, solid cortical bone in proximity of blood vessel. (A3, A4, A5) Diaphyseal cortical bone in control mice was free of macro-pores. (B) Morphology of Nf1Prx1 humeri. (B1) Note a broadened and inhomogeneously mineralized tuberosity with rough mineralization boundaries and increased porosity. (B2) A massively enlarged nutrient artery traversing site and (B3) a large ectopic lesions were observed in the cortical bone of Nf1Prx1 mice. (B4, B5) von Kossa/Masson Goldner histology of the diaphyseal bone cortex in blood vessel proximity. Note, unmineralized bone matrix (osteoid) surrounding the centrally localized blood vessel. (C) Nf1Col1 mouse humeri. (C1) Mutant deltoid tuberosity is widened with inhomogeneous mineral distribution and uneven bone boundaries. (C2-3) The size of the nutrient artery traversing site within diaphyseal bone cortex was normal in Nf1Col1 mice. (C4-5) In Nf1Col1 cortical bone small mineralization defects show a narrow osteoid rim surrounding blood vessels. These changes were of much smaller size compared to the Nf1Prx1 model. (D) Histomorphometric analysis of humerus cortical bone in the region E2 of Nf1Prx1 mice. Increased relative unmineralized bone tissue area (O.Ar/B.Ar) and blood vessel area (BlVes.Ar/B.Ar) (control n = 3, Nf1Prx1 n = 5). (E) Volume and number of mineralization defects (lacunae range 0.05–1*106 µm3) in Nf1Prx1 cortical bone (region E2). High-resolution scans were evaluated with CTan (skyscan) software. Increased relative cortical bone porosity (summed lacunae volumes per cortical bone volume, Lc.V/Ct.BV) as well as lacunae number (Lc.N/Ct.BV) (control and Nf1Prx1 n = 5) in Nf1Prx1 humeri. Statistical significance - t-test, ** p≤0.001. (F) Immunostaining with pan-endothelial antibody confirms presence of blood vessels within mineralization defects in Nf1Prx1 cortical bone. (G) Nf1Prx1 cortical bone in the region E2, von Kossa/Masson-Goldner histology showing mineralization defects through developmental stages P14, P35 and P49. Scale bars - 50 µm. Abbreviations: blood vessels (bv), bone (b), bone marrow (bm), osteoid (o).
Figure 2.
Diminished organic matrix properties in Nf1Prx1 mice.
(A) Picrosirius red stained bone sections imaged with polarized light. Homogenous red staining in controls indicates highly packed and thick collagen. Heterogeneous red-yellow-green staining of Nf1Prx1 bone sections is indicative of diminished packaging and thickness of bone collagen. Note, there is no picrosirius red staining within non-mineralized bone tissue (osteoid) surrounding blood vessels. Scale bar shows 20 µm. (B) Silver staining (AgNOR) detects the osteocytic network and other accumulations of acidic matrix proteins. Note the large unstained area around blood vessels in Nf1Prx1 humerus. Scale bar shows 50 µm. (C) Sections of humerus cortex stained with Toluidin and Safranin O. Toluidin stained osteocyte (Ot.) appear blue with dark blue nuclei (black arrowhead). Presence of nuclei suggests vitality of cells in the bone cortex. Osteoid (dotted line) near to the blood vessel was stained light blue (Toluidin) or light red (Safranin O). Light blue or red staining indicates that bone lesions are not composed of cartilaginous matrix, which with these methods stains purple (Toluidin) or dark red (Safranin O) (not shown). Ot. in non-mineralized areas are viable (nuclei marked with grey arrow head). Scale bar represents 50 µm. Abbreviations: blood vessels (bv), bone (b), bone marrow (bm), osteoid (o).
Figure 3.
Nf1Prx1 bone tissue shows diminished mechanical strength and increased micro-porosity due to increased osteocyte lacuna size.
(A) Diagram of the analytical setup used for tensile experiment. Measurements were done on laser dissected cortical bone slices from P90 mice. (B) A typical tensile stress-strain pattern in controls showed a linear elastic modulus (E-modulus) phase that is followed by the yield point plateau and the ultimate stress point. Note, increased elasticity and diminished ultimate strength in bone slices of Nf1Prx1 humerus compared to controls. (C) Diminished E-modulus (slope of tensile curve) and (D) ultimate stress point (point of maximal load before tissue failure) in bone tissue slices from 3 months old Nf1Prx1 (n = 14) and control (n = 15) mice. (E) Quantitative microCT analysis of the total (summed) lacunae porosity (Lc.V/BV) within the lacunae range from 100-4000 µm3. Note the significant increase of Lc.V/BV in Nf1Col1 and Nf1Prx1 mice. (F) The summed volume of Ot. lacunae (Lc.V) within the size range 100–4000 µm3 with an increment of 100 µm3 (histogram). Note the shift of summed Lc.V fractions towards higher volume range in Nf1Col1 and Nf1Prx1 mutants. Insert (a) - Nf1Col1 mice demonstrated reduced summed Lc. volume in the intervals 200–300 and 300–400 µm3 and (b) - summed Lc.V in fractions 800–900 and 900–1000 µm3. Insert (c) - Nf1Prx1 mice showed reduced summed Lc. volume in the intervals 100–200 and 200–300 µm3 and (d) - summed Lc.V in fractions 700–800 and 800–900 µm3. (G) The total number of Ot. lacunae (Lc.N) was unaffected in both mouse models. (H) Ot. lacunae morphology (range from 100–4000 µm3) assessed by measuring the x, y and z axis length in a selected volume of ROI E2. Note, lacunae size is increased in all dimensions in Nf1Col1 and Nf1Prx1 mice. The number of analyzed bone samples: Nf1Prx1 n = 5, controls n = 5; Nf1Col1 n = 3, controls n = 3. Statistical analysis was performed with unpaired Student t-test, * p≤0.05 ** p≤0.01.
Figure 4.
Diminished bone stiffness, bone mineralization and organic matrix formation in Nf1Prx1 mice.
(A) Scanning acoustic microscopy (SAM) was applied to show elastic properties of the bone matrix. SAM measurements were performed within six ROIs E1a–E3b on anterior (a) and posterior (b) bone cortex. Images illustrate impedance values (Z in MRayl) according to the color scale (left). Nf1Prx1 mutants show diminished impedance values within all analyzed ROIs. (B) Quantitative evaluation of SAM measurements reveals decreased Z values in Nf1Prx1 humerus in all analyzed ROIs (control n = 4, Nf1Prx1 n = 4). Nf1Col1 humeri did not demonstrate diminished Z values (control n = 5, Nf1Col1 n = 4). (C) The cross sections of humerus at the level E2 with color coded BMD values measured by phantom calibrated microCT. The yellow and green coloring indicates low and high BMD values, respectively. Scale bar - 600 µm. (D) Cortical bone mineral density (BMD) was assessed by microCT within region E1–E3 in humeri of Nf1Prx1 (control n = 5, Nf1Prx1 n = 5) mice. BMD was reduced in the Nf1Prx1 model in all ROIs (for definition see Fig.S1). Statistical significance - t-test, * p≤0.05 and ** p≤0.01.
Figure 5.
Cortical bone in NF1 tibial dysplasia is characterized by heterogeneous mineralization, diminished collagen thickness and increased micro-porosity.
(A) MicroCT analysis of human cortical bone samples (tibia). Bone mineral density was calibrated according to Houndsfield units (HU) and color coded (see HU scale). HU values measured in ROI1 (cortical bone without blood vessels) and ROI2 (cortical bone near blood vessel) were in control specimen similar. However, in the NF1 bone sample HU values were in proximity of large vessels decreased (ROI2). The analyzed bone sample was obtained pre-fracture from an individual affected by NF1 with tibial bowing. (B) Two further mineralized bone samples were obtained from cortical bone of NF1 individuals with pseudarthrosis. Samples were analyzed by picrosirius red histology and imaged in polarized light (large image) and bright light (inset). Collagen fibers follow osteonal organization in control specimens. Note that in the NF1 cortical bone sample collagen fiber organization appears less orderly with abundant thin (green) collagen fibers. (C) Ot. morphology was visualised with AgNOR staining. Ot. are spindle shaped (inset) and regularly distributed in control cortical bone. In contrast, Ot. are round and irregularly distributed in NF1 cortical bone. (D) Histomorphometry of AgNOR stained bone sections showing: relative Ot. area (Ot.Ar/B.Ar), Ot. number per bone area (Ot.N/B.Ar), individual Ot. area (Ot.Ar) and individual Ot. circumference (Ot.Cir). Presented data are mean values with standard deviations (control n = 3, NF1 n = 2). (E) Volumetric microCT analysis showing increased specific lacunae (Ot.) volume (Lc.Vol) and surface (Lc.Sur) in NF1 tibial dysplasia cortical bone as compared to controls. Statistical analysis was performed with unpaired t-test, ** p≤0.01. Abbreviations: blood vessels (bv) and bone (b). All scale bars represent 50 µm.
Figure 6.
Neurofibromin is a critical regulator of cortical bone integrity and function.
(A) Ablation of Nf1 in pre-osteoblasts (Nf1Col1) results in low bone mass phenotype with hyperosteoidosis. Loss of Nf1 in mesenchymal progenitor cells (Nf1Prx1) produces a complex phenotype characterized by low bone mass, hyperosteoidosis, increased micro-porosity (Ot.), macro-porotic mineralization lesions, and persistence of blood vessels. Moreover, loss of neurofibromin causes defective inorganic and organic bone matrix formation especially in proximity of blood vessels. (B) Micro- and macro-porosity contributes differentially to overall structural destabilisation in NF1 bone. In controls micro- (Ot.) and macro-porosity (blood vessels) encompasses approx. 2% and 0.2% of cortical bone volume, respectively. In Nf1Prx1 mutant bone micro-porosity is elevated to approx. 3.5%. Importantly macro-porosity is amplified to 1.0% due to blood vessel persistence and bone mineral lesions.