40 Genetically Modified Models for Bone Repair
Delayed fracture repair and nonunions represent a major clinical challenge due to the lack of understanding of the causes for skeletal repair defects and limited approaches to correct these defects. Basic research in bone biology has had major impacts on the field of orthopedic surgery. The discovery of Bone Morphogenetic Proteins (BMPs) and the purification of BMP7 and BMP2 revolutionized the field of orthopedic research and led to new treatments of long bone fractures and spinal fusion. 1 Bone autografts or allografts are largely used worldwide, yet stem cell–based approaches are not yet generalized, although several clinical trials are under way to test the efficacy of bone marrow–derived mesenchymal stromal cells. There are many efforts under way in numerous biology and bioengineering research laboratories to better understand the cellular and molecular bases of skeletal regeneration. These efforts are based on in vitro approaches to improve the conditions for stem cell transplantation, or to define the molecular regulation of cell differentiation in the osteogenic and/or chondrogenic lineages, the two cell lineages that produce the bone and cartilage matrices indispensable for fracture consolidation. In parallel, in vivo approaches are required to place these in vitro data back in the context of endogenous bone repair, which cannot be entirely modeled in vitro due to the numerous cell types involved, and the complex interplay among these cell types and the supporting vasculature. Therefore, animal models, and more specifically genetically modified mouse models, are essential to define the roles of specific cell types and molecular pathways, and to test new therapies prior to their application in humans.
Jargon Simplified: Genetically Modified Mouse Model
A mouse whose genetic material (deoxyribonucleic acid [DNA]) has been modified using genetic engineering tools. Genetic modifications may include mutation, insertion, or deletion of genes.
40.1 Advantages of Mouse Models to Study Bone Repair
Although large animal models are essential to test new orthopedic devices and for clinical trials due to their size and anatomy closer to human, smaller animal models and in particular mice have become very popular in basic orthopedic research. Mice are more cost-effective and have a shorter gestation time than large animal models. Many physiological processes, including bone repair, are accelerated but share common features between mice and human, justifying the use of mouse models to study diseases and to test the efficacy of new drugs for disease conditions like cardiovascular diseases, neurological disorders, diabetes, and cancer. In the skeletal system, mouse models are employed to elucidate the genetic bases of rare bone diseases, and the mechanisms responsible for osteoarthritis, osteoporosis, or skeletal repair.
The stages of bone repair can be described in both human and mouse in four phases beginning with the inflammatory phase, followed by soft callus formation, hard callus formation, and the remodeling phase. Careful analyses of the cellular and molecular processes regulating these stages of repair have revealed that these processes can be extrapolated from mouse to human.
Jargon Simplified: Genetic Modification Terms
Genetic Screen: Large-scale mutagenesis (via exposure to irradiation, mutagens, or random DNA insertion into the genome) of an animal population followed by phenotypic analyses and genotyping in order to identify new genes involved in a given biological function (for example genes involved in bone formation).
Transgenesis: Alteration of the genome of an organism via insertion of an exogenous gene, called a transgene, into the genomic DNA. This transgene is then transmitted to the organism offspring.
Targeted Gene Deletion (KO Mouse Model): Deletion of a specific gene in the whole organism, which can be achieved by deleting either the entire gene sequence on the chromosome or a small portion of the gene (leading to misexpression or interruption of the gene sequence).
Conditional KO Mouse Models: Mouse carrying a targeted gene deletion in a cell-, tissue-, or organ-specific manner, so that only one cell type, tissue, or organ is affected by the mutation (the Cre-Lox system is the most common tool used to generate conditional mouse KO models).
Inducible Gene Inactivation (Inducible KO): Targeted gene deletion at a given time point (for example at the time of bone injury) in order to bypass the effects of the mutation during embryonic development. The Cre-Lox system allows both conditional and inducible gene inactivation, thanks to tamoxifen-inducible CreER. Following tamoxifen injection into the mouse, CRE recombinase is expressed and causes gene inactivation in the tissue/cell of interest.
Over the past decade, mouse genetics has allowed scientists to identify key genes involved in embryonic bone development based on genetic screens and has provided the ability to mutate specific genes in the genome to study the phenotypic consequences of these mutations. This genetic research is directly relevant to human skeletal development and diseases as mouse and human genomes share 95% homology. These advances have been possible thanks to various technologies such as transgenesis and the ability to culture embryonic stem cells for targeted gene deletion via homologous DNA recombination (knockout [KO] mouse models). More elaborated genetic technologies have been subsequently developed to induce specific mutations in particular cell types or tissues (conditional KO mouse models), and have even offered the possibility to induce mutations at a particular time during the life of the genetically modified animal (inducible gene inactivation). Combined with reporter mouse models, these tools also allow gene expression analyses and cell lineage tracing in vivo. Following are detailed descriptions of these various mouse models illustrated by specific examples of orthopedic applications.
40.2 Transgenic and Reporter Mouse Models
Using transgenesis, DNA encoding a specific gene sequence under its own regulatory gene sequence or another promoter can be introduced directly into the fertilized egg via microinjection and is inserted randomly into the mouse genome. The injected eggs are then implanted into a pseudo-pregnant female, which will generate mice carrying the inserted DNA sequence in every cell of the organism and capable of transmitting this DNA sequence to the next generation. Using this technique, a given gene can be overexpressed in a specific cell type if placed under the control of a specific promoter of this cell type such as collagen type 2 promoter for chondrocyte, collagen type 1 promoter for osteoblasts, and tartrate-resistant acid phosphatase promoter for osteoclasts. 2 The gene of interest can be a reporter gene such as LacZ (encoding beta-galactosidase) or green fluorescent protein expressed under a cell- or tissue-specific promoter to visualize gene expression on tissue sections. Chen et al 3 reported the expression of the LacZ transgene controlled by a c-fos minimal promoter and TCF-binding motifs to show the spatial and temporal activation of the WNT pathway, which is essential for bone formation and fracture repair. Using a combination of various reporter genes, several specific cell types can be visualized simultaneously on a given tissue section of the fracture callus to better describe the interrelations of osteoblasts, chondrocytes, endothelial cells, and osteoclasts during bone repair. 4
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