Local low-dose lovastatin delivery improves the bone-healing defect caused by Nf1 loss of function in osteoblasts - PubMed (original) (raw)

Local low-dose lovastatin delivery improves the bone-healing defect caused by Nf1 loss of function in osteoblasts

Weixi Wang et al. J Bone Miner Res. 2010 Jul.

Abstract

Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the Nf1(ob) (-/-) conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21- to 28-day postfracture period, callus volume, as expected, decreased in wild-type but not in Nf1(ob) (-/-) mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro-computed tomography were decreased in Nf1(ob) (-/-) callus-bridging cortices and trabecular compartments compared with wild-type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in Nf1(ob) (-/-) calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfbeta. Callus strength measured by three-point bending 28 days after fracture was reduced in Nf1(ob) (-/-) versus wild-type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low-dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low-dose lovastatin may improve bone healing in NF1 patients.

2010 American Society for Bone and Mineral Research.

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Figures

Fig. 1

Lack of Nf1 specifically in osteoblasts delays bone healing. Callus longitudinal X-ray analyses in WT and formula image mice. Callus volume clearly decreased in the late stages of bone healing (days 21 to 28) in WT but not in mice.

Fig. 2

Callus trabecular and cortical volumetric parameters are affected by lack of Nf1 in osteoblasts. (A, B) 3D µCT representative images from WT and formula image calluses on days 21 (A) and 28 (B) after fracture. Callus size was increased in mutant mice, whereas the amount of callus and bridging cortices calcified bone was decreased. Side view (top panel), cross view (middle panel), and bridging periosteum shell (bottom panel). (C) 3D µCT quantification of callus tissue volume (TV), callus bone volume over total volume (BV/TV), and bridging cortices BV/TV in WT and formula image mice 21 and 28 days after fracture (*p < .05, n = 10 to 12 mice/group).

Fig. 3

Lack of Nf1 in osteoblasts weakens callus mechanical properties. (A) Callus strength (maximum force) as measured by three-point bending 28 days after fracture was significantly reduced in formula image calluses. (B) Callus stiffness was reduced in mice, but the difference between mutant and WT controls did not reach significance (NS) (*p < .05, n = 10 to 12 mice/group).

Fig. 4

Increased osteoclast surface, osteoid, and number of cartilaginous remnants in formula image calluses. (A) TRAP+ osteoclast surface (OcS) and bone surface (BS) were significantly increased in calluses 28 days after fracture compared with WT calluses (TRAP-stained and hematoxylin-counterstained). Osteoclast-free osteoid surfaces are underlined with a doted line. (B) Osteoid volume over bone volume (OVcallus/BVcallus) was significantly increased in formula image calluses compared with WT calluses (von Kossa/van Gieson staining) 28 days after fracture. (C) The number of cartilaginous remnants was significantly increased in calluses compared with WT calluses 21 days after fracture (representative image of section stained with safranin-O and counterstained with methyl green) (*p < .05, n = 7 to 12 mice/group).

Fig. 5

Gene expression changes induced by Nf1 loss of function in osteoblasts. (A) Rankl, Opn, and Tgfβ but not Bmp2 mRNA expressions were significantly increased 14 days after fracture in formula image calluses compared with WT calluses. Gene expression was measured by quantitative TaqMan qRT-PCR and normalized by the expression of 18S rRNA (*p < .05, n = 3 to 4 calluses/group).

Fig. 6

Lovastatin corrects ERK1/2 activation in _Nf1_−/− osteoblasts. In vitro Nf1 recombination by cre-adenovirus (+) (cre)-infection of _Nf1_flox/flox osteoblasts increased ERK1/2 phosphorylation compared with mock (−) adenovirus-infection (bottom panel, representative blot, n = 3). Lovastatin (LOVA) treatment blunted this effect in cre-adenovirus (cre)-infected _Nf1_flox/flox osteoblasts. The ratio P-ERK:ERK is quantified in the top panel.

Fig. 7

Lovastatin improves bone healing in formula image mice. (A) Lovastatin release profile. Cumulative release reached 100% within 8 days. (B) 3D µCT quantification of callus tissue volume (TV), callus bone volume over total volume (BV/TV), and callus bridging cortices BV/TV 28 days after fracture in mice treated with lovastatin (LOVA) or control microparticles. (C) Decreased callus osteoid volume over total volume (OVcallus/TVcallus) in formula image mice treated by lovastatin. (D) Lovastatin increased maximum force in mice compared with control-treated mice to an extent that this mechanical property was no longer different between WT control-treated mice and lovastatin-treated mice (*p < .05, n = 10 to 12 mice/group). (E) Tgfβ mRNA expression was decreased significantly by lovastatin 14 days after fracture in formula image calluses. Gene expression was normalized by the expression of 18S rRNA (*p < .05, n = 3 to 4 per group).

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Local low-dose lovastatin delivery improves the bone-healing defect caused by Nf1 loss of function in osteoblasts - PubMed (original) (raw)