Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

Abstract

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.

Epigenetics in the perioperative period

“…Using bone healing as experimental paradigm, Bais and colleagues demonstrated the complex temporal sequence of transcriptome changes during healing, involving more than 300 genes (Bais et al, 2009). This confirms previous investigations that demonstrate the involvement of epigenetic mechanisms in the induction of wound healing (Shaw and Martin, 2009).…”

“…Opiate receptors ↔ development (Hwang et al, 2007) Local anaesthetics ↓ methylation (Lirk et al, 2012(Lirk et al, , 2014 Opiates ↑ methylation (Doehring et al, 2013) Opiate-induced hyperalgesia (Doehring et al, 2013) ↓ MCP3 expression → pain (Imai et al, 2013) ↓ GAD65 expression → pain (Zhang et al, 2013) Prefrontal cortex methylation (Tajerian et al, 2013) TRPA1 methylation ↔ pain (Bell et al, 2014) Transcriptome in inflammation (Bais et al, 2009) Transcriptome in healing (Kruidenier et al, 2012;Barnes et al, 2013;Belkina et al, 2013) Cell migration/prolif, angiogenesis (Rafehi et al, 2011) Heritable response to injury (Zeybel et al, 2012) Wound healing inflammation…”

Gene Therapy Applications for Fracture Repair

“…Fracture repair involves the interplay of the well regulated interdependent processes of chondrogenesis and osteogenesis. These processes have been characterized by gene profiling of the regulated expression of numerous growth factors, growth factor receptors, cytokines and transcription factors (Hadjiargyrou et al, 2002;Li et al, 2005;Rundle et al, 2006;Bais et al, 2009). The effect of growth factors and other regulatory proteins on fracture www.intechopen.com repair have also been characterized by observational studies of fracture repair in normal animal subjects compared to those with genetically defined deficiencies, i.e., knockout mice.…”

“…This concept is stated as "repair recapitulates development" and has been associated with bone, as it has with other tissues (Gerstenfeld et al, 2003;Bais et al, 2009). It is highly informative to view fracture repair in the context of the cells that mediate the development of the different types of bone during embryogenesis: osteoblast maturation for intramembranous bone repair and chondrocyte maturation and osteoblast maturation for endochondral bone repair.…”

“…The skeletal gene therapy strategies to date have targeted the osteoblasts, osteogenic progenitors, chrondrogenic progenitors and other cells within the fracture repair region. To begin to identify appropriate target genes that might be used to accelerate healing, several laboratories subjected mRNA isolated from different aspects of fracture repair to gene profiling procedures to gain insight to the molecular pathways regulating each situation, including the stages of fracture repair (Hadjiargyrou et al, 2002;Li et al, 2005;Rundle et al, 2006) and the expression of genes of current therapeutic interest, such as stem cell genes (Bais et al, 2009). A variety of genes, involved in activation and repression, were discovered to participate in each stage of the repair process and suggest therapeutic candidates for fracture repair, but the first target genes selected for fracture therapy were those that influenced the rate of osteoblast differentiation and thus bone formation (Fig.…”