Modeling bone morphogenetic protein and bisphosphonate combination therapy in wild‐type and Nf1 haploinsufficient mice

Abstract

Recombinant bone morphogenetic proteins (BMPs) show promise in treating the orthopedic complications associated with neurofibromatosis type 1 (NF1), such as congenital pseudarthrosis of the tibia. Minimal scientific information regarding the effects of BMP in the context of NF1 is available. As abnormalities in both bone formation and resorption have been documented in Nf1-deficient mice, we hypothesized that inadequate BMP-induced bone formation could be augmented by cotreatment with the bisphosphonate zoledronic acid (ZA). First, primary osteoblasts isolated from wild type (Nf1) mice were cultured in the presence and absence of BMP-2. While Nf1 þ/À cells exhibited less osteogenic potential than Nf1cells, alkaline phosphatase expression and matrix mineralization for both genotypes were enhanced by BMP-2 treatment. To model this response in vivo, 20 mg BMP-2 was implanted intramuscularly into the quadriceps of mice to induce heterotopic bone. Radiographs revealed significantly less net bone formation in Nf1 þ/À mice compared to Nf1 þ/þ controls. To test the effect of an antiresorptive agent, mice were cotreated twice weekly from postoperative day 3 with 0.02 mg/kg ZA or with saline. ZA treatment led to a synergistic increase in the amount of heterotopic bone in both Nf1 þ/þ and Nf1 þ/À mice compared with saline controls, as measured by DEXA and histomorphometry. Thus, the anabolic deficiency noted in Nf1 þ/À mice is amenable to stimulation by BMP-2, but mineralized tissue formation remains below that of Nf1 þ/þ controls. Bisphosphonate combination therapy is superior to BMP therapy alone in terms of net bone production in vivo in both wild-type and Nf1-deficient mice. ß

Biologic Characteristics of Fibrous Hamartoma from Congenital Pseudarthrosis of the Tibia Associated with Neurofibromatosis Type 1

“…Nf1 haploinsufficient mice showed a lower number of mesenchymally originated cells with the osteoblastic phenotype 13 and premature apoptosis, higher proliferation, and attenuated differentiation of committed osteoprogenitors 14 . Moreover, Schindeler et al showed reduced efficacy of surgically implanted rhBMP-2 22 . Therefore, the Nf1 ± genotype could contribute to fibrous hamartoma development by blocking the osteoblastic differentiation of periosteal cells.…”

“…Schindeler et al 22 showed that the addition of bisphosphonate improved deficient ectopic bone formation by BMP-2 in Nf1± mice. The suppression of osteoclasts is also expected to prevent fibrous hamartoma tissue from encroaching into tibial bone tissue and thus help osteosynthesis or prevent recurrence of fracture.…”

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

“…(10) Heterozygous Nf1 þ/À deficient mice exhibited an impaired response in BMP-7 in an ectopic bone formation assay (11) as well as reduced bone healing in a distal tibial fracture model. (12) More advanced models have been subsequently generated in an attempt to model the double inactivation of NF1 seen in some human bone defects.…”

“…Adjacent sections were stained for tartrate-resistant acid phosphatase (TRAP) expression to highlight osteoclasts, and counterstained with 0.4% Light Green. (11) Sections were also stained for BrdU using a commercial kit (Invitrogen). Stained sections were quantified and analyzed by a blinded assessor using the Bioquant Analysis System.…”

Skeletal abnormalities in neurofibromatosis type 1: Approaches to therapeutic options

“…A recent study demonstrated that the combination of bisphosphonates and bone morphogenetic protein 2 (BMP2, a protein which induces bone and cartilage formation) improved net bone production by Nf1 þ/À cells in an in vivo model of heterotopic bone formation [Schindeler et al, 2008b]. This treatment did not attempt to correct the specific signaling defects associated with Nf1 À/À or þ/À osteoblasts, but represented an initial approach.…”