Rodent models of joint injury are vital for the development of next-generation treatment strategies to prevent post-traumatic osteoarthritis (PTOA). In recent decades, the application of genetic engineering techniques to the well-characterized genome of mice has resulted in the revolutionary development of transgenic and knockout strains. While these mouse models are now a fundamental tool in orthopaedic research, similar rat models have been lagging. However, an improving knowledge of the rat genome combined with continued advances in genome editing now make transgenic rat development more practical. There are important differences between rats and mice that make the latter species preferred for initial testing of novel therapeutics in vivo. For example, the ra is routinely used for evaluating new stem cell-based strategies to stimulate osteochondral tissue repair, whereas mouse joints are too small for this purpose. Rat models of injury have been advanced by the ability to noninvasively image stem cells genetically engineered to express molecular reporters. Among the available reporters, the luciferases catalyze intense bioluminescent reactions that can be measured quantitatively with established imaging platforms. If rats were genetically engineered to express a luciferase specifically within cells committed to the chondrogenic lineage, this would allow noninvasive evaluation of endogenous chondroprogenitor cell (CPC) differentiation. CPC chondrogenesis within cartilage and meniscal defects is an important bottleneck to repair, and this rat model would help test strategies to overcome this bottleneck. The first project aim will be to generate a transgenic rat in which Firefly luciferase (FLuc) and LacZ are expressed specifically by cells committed to the chondrogenic lineage, while Renilla luciferase (RLuc) and inducible CreERT2 recombinase are expressed constitutively by all cells. Exploiting the postnatal specificity of type II collagen expression within cells of the chondrogenic lineage, we will use regulatory sequences from the Col2a1 gene for controlling FLuc and LacZ expression. Once the dual- lucifase trasngenic rat has been made, the second project aim will be to demonstrate its utility using two in vivo models of joint injury: (i) osteochondral defects made in wild type rats receiving transgenic CPCs, and (ii) a meniscectomy model of PTOA in wild type rats, injecting transgenic CPCs. For these initial studies, mesenchymal stem cells from bone marrow and synovium will be compared. Once the transgenic rat has been characterized, it will be made available as a resource to the orthopaedic research community. When combined with existing bioluminescence assays and imaging platforms, this strain will permit sensitive, quantitative measuring of pro-chondrogenic activity i studies of not only joint injury but also endochondral bone formation (e.g., fracture healing). The engineering of tamoxifen-inducible CreERT2 into this rat will allow it to be crossed with future LoxP rats for gain and loss of function studies, greatly broadening its application and, ultimately accelerating orthopaedic research.
Management of cartilage damage within synovial joints remains a clinical challenge. The animal model proposed here will help develop new strategies for stimulating cartilage repair by permitting noninvasive assessment of cartilage progenitor cell differentiation, an important bottleneck in the healing process.
| Ferreira, E; Porter, R M (2018) Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 7:263-273 |
