Temporal Gene Expression Profiling during Rat Femoral Marrow Ablation-Induced Intramembranous Bone Regeneration

Abstract

Enhanced understanding of differential gene expression and biological pathways associated with distinct phases of intramembranous bone regeneration following femoral marrow ablation surgery will improve future advancements regarding osseointegration of joint replacement implants, biomaterials design, and bone tissue engineering. A rat femoral marrow ablation model was performed and genome-wide microarray data were obtained from samples at 1, 3, 5, 7, 10, 14, 28, and 56 days post-ablation, with intact bones serving as controls at Day 0. Bayesian model-based clustering produced eight distinct groups amongst 9,062 significant gene probe sets based on similar temporal expression profiles, which were further categorized into three major temporal classes of increased, variable, and decreased expression. Osteoblastic- and osteoclastic-associated genes were found to be significantly expressed within the increased expression groups. Chondrogenesis was not detected histologically. Adipogenic marker genes were found within variable/decreased expression groups, emphasizing that adipogenesis was inhibited during osteogenesis. Differential biological processes and pathways associated with each major temporal group were identified, and significantly expressed genes involved were visually represented by heat maps. It was determined that the increased expression group exclusively contains genes involved in pathways for matrix metalloproteinases (MMPs), Wnt signaling, TGF-β signaling, and inflammatory pathways. Only the variable expression group contains genes associated with glycolysis and gluconeogenesis, the notch signaling pathway, natural killer cell mediated cytotoxicity, and the B cell receptor signaling pathway. The decreased group exclusively consists of genes involved in heme biosynthesis, the p53 signaling pathway, and the hematopoietic cell lineage. Significant biological pathways and transcription factors expressed at each time point post-ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

Adult Stem Cell Mobilization Enhances Intramembranous Bone Regeneration: A Pilot Study

“…AMD3100 (plerixafor) is FDA approved for use in patients in combination with granulocyte colony-stimulating factor to mobilize HSCs. The antibodies used for flow cytometry ( For marrow ablation surgery, animals were prepared and underwent surgery as described previously [58]. The marrow cavity of the left femur was accessed using a 25-gauge needle, mechanically ablated, and subsequently flushed with a 30-gauge needle and 0.1 mL sterile saline.…”

“…Marrow ablation induces intramembranous bone regeneration by physically removing native bone and inducing a repair response with defined histologic phases of healing and temporal patterns of gene expression [58] and provides relatively quick end points for assessing bone repair. However, the potential effects of AMD3100-induced stem cell mobilization on intramembranous bone regeneration after marrow ablation is unclear.…”

Intramembranous bone regeneration and implant placement using mechanical femoral marrow ablation: rodent models

“…There are several bone repair models currently in use, including fracture healing, 1,2 marrow ablation, [3][4][5][6][7][8][9] calvarial defect, 10,11 distraction osteogenesis, 8,12 segmental replacement 13 and spinal fusion. 14,15 Model choice depends upon the type of bone formation to be studied, as well as the importance of loadbearing, skeletal region and ability to translate to the clinic.…”

“…14,15 Model choice depends upon the type of bone formation to be studied, as well as the importance of loadbearing, skeletal region and ability to translate to the clinic. The marrow ablation model is primarily used for two main purposes: (1) as a model for intramembranous bone regeneration 3,4,16,17 and (2) as a model to study implant fixation in orthopedics 9,[18][19][20][21] and dentistry. 22,23 Therefore, the model is of interest to orthopedic and dental research, including biomaterial specialists, implant designers and molecular biologists as well as the field of regenerative medicine.…”

“…9,25,26 Like nearly all wound repair responses, intramembranous bone formation following marrow ablation has inflammatory, repair and remodeling phases each of which are time-dependent, characterized histologically and genetically. 4 These phases for intramembranous bone formation are distinct from endochondral-based fracture repair. For instance, bone formation following marrow ablation, unlike endochondral-based fracture repair, does not include chondrocytes and lacks collagen type II mRNA expression.…”