Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase/nucleoticle pyrophosphatase phosphodiesterase 1 double-deficient mice

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2(-/-)) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp(-/-) mice is corrected in [Akp2(-/-); Enpp1(-/-)] double-knockout mice. Here we have examined the appendicular skeletons of Akp2(-/-), Enpp1(-/-), and [Akp2(-/-);Enpp1-(/-)] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2(-/-), and [Akp2(-/-); Enpp1(-/-)] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2(-/-) but also in Enpp1(-/-) and [Akp2(-/-); Enpp1(-/-)] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1(-/-) calvarial osteoblasts but decreased mineral deposition by Enpp1(-/-) long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2(-/-) mice and the hypomineralized phenotype of the long bones of Enpp1(-/-) mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.