The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD+ deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cells of the limbs. At birth, NamptΔPrx1 exhibit dramatic limb shortening due to death of growth plate chondrocytes. Administration of the NAD precursor nicotinamide riboside during pregnancy prevents the majority of in utero defects. Depletion of NAD post-birth also promotes chondrocyte death, preventing further endochondral ossification and joint development. In contrast, osteoblast formation still occurs in knockout mice, in line with distinctly different microenvironments and reliance on redox reactions between chondrocytes and osteoblasts. These findings define a critical role for cell-autonomous NAD homeostasis during endochondral bone formation.

A Transgenic Rat for Noninvasive Assessment of Chondrogenesis in Vivo.

OBJECTIVE: To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. DESIGN: A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. RESULTS: Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. CONCLUSIONS: This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.