Microstructure and homogeneity of distribution of mineralised struts determine callus strength.

Non-invasive assessment of fracture healing, both in clinical and animal studies, has gained favour as surrogate measure to estimate regain of mechanical function. Micro-computed tomography (µCT) parameters such as fracture callus volume and mineralisation have been used to estimate callus mechanical competence. However, no in-depth information has been reported on microstructural parameters in estimating callus mechanical competence. The goal of this study is to use differently conditioned mice exhibiting good and impaired fracture healing outcomes and investigate the relationship between µCT imaging parameters (volume, mineralisation, and microstructure) that best estimate the callus strength and stiffness as it develops over time. A total of 99 mice with femoral fracture and intramedullary stabilisation were divided into four groups according to conditioning: wild type, NF1 knock-out, RAG1 knock-out and macrophage depleted. Animals were sacrificed at 14, 21, 28 or 35 days and µCT parameters and torsional stiffness and strength were assessed post-sacrifice. Using linear regression for all groups and time points together, torsional stiffness could be estimated with strut thickness, strut number and strut homogeneity (R² = 0.546, p < 0.0001); torsional strength could be estimated using bone mineral density, strut thickness and strut homogeneity (R² = 0.568, p < 0.0001). Differently conditioned mice that result in different fracture healing outcomes have been shown to result in varying structural, material and volumetric µCT parameters which can be used to estimate regain of bone strength. This study is the first to demonstrate that microstructure and strut homogeneity influence callus stiffness and strength.

Deterioration of fracture healing in the mouse model of NF1 long bone dysplasia.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 manifests as a heritable susceptibility to tumours of neural tissue mainly located in the skin (neurofibromas) and pigmented skin lesions. Besides these more common clinical manifestations, many NF1 patients (50%) have abnormalities of the skeleton. Long bones are often affected (usually the tibia) and the clinical signs range from bowing to spontaneous fractures and non-unions. Here we present the analysis of bone fracture healing in the Nf1(Prx1)-knock-out mouse, a model of NF1 long bone dysplasia. In line with previously reported cortical bone injury results, fracture healing was impaired in Nf1(Prx1) mice. We showed that the defective fracture healing in Nf1(Prx1) mice is characterized by diminished cartilaginous callus formation and a thickening of the periosteal bone. These changes are paralleled by fibrous tissue accumulation within the fracture site. We identify a population of fibrous tissue cells within the Nf1 deficient fracture as alpha-smooth muscle actin positive myofibroblasts. Additionally, histological and in-situ hybridization analysis reveal a direct contact of the fracture site with muscle fascia, suggesting a possible involvement of muscle derived cells in the fracture deterioration.