Expression of P2X4R, P2X7R and P2Y2R in fracture healing: An in vivo study on purinergic signalling in fracture healing.

Subash Suntharalingam

Student thesis: Master thesis

Abstract

Bone fractures are associated with morbidity and mortality and constitutes a major economic burden for societies. It is estimated that 43.300 lives were lost as a result of incident fractures in EU in 2010. In recent years, there has been an increasing appreciation of the therapeutic potentials of purinergic signalling as a toolkit for tissue repair. Nucleotides are released into the extracellular environment due to tissue stress, and here they are found to serve as a damage signal by acting on purinergic receptors. Purinergic receptors are found on the cell surface of almost all cell types where they propagate downstream signalling. Although purinergic signalling has been linked to very central roles in bone biology, such as differentiation of mesenchymal and hematopoietic stem cell into osteoblast and osteoclast, bone and cartilage mineralisation and biomechanical properties, the involvement of P2 receptors in fracture healing remains to be elucidated. We investigated the association of P2X4R, P2X7R and P2Y2R gene expression to fracture healing. A closed unilateral fracture model of mid-diaphysal tibia, was applied to 7 month old female Sprague Dawley (SD) rats. The rats were terminated for expression analysis using RT-PCR on day 1, 2, 3, 7 and 21 and for bone property analysis by x-ray and Dual-energy x-ray absorptiometry on day 7 and 21. External callus formation and mineralisation was seen on day 21 by x-ray and DEXA. BMD was increased from 0.165 g/cm2 on day 7 to 0.182 g/cm2 on day 21 (p=0.021, fig 12 B). BMC was increase from 0.060 g on day 7 to 0.106 g on day 21 (p=0.021, fig 12 C). Bone area was increased from 0.378 mm2 on day 7, to 0.565 mm2 on day 21 (p=0.021, fig 12 D).
RNA extraction from bone is complicated by the hard nature of bone tissue. A protocol for homogenization and RNA extraction from bone tissue was successfully developed. Bone tissue was homogenized and lysed using a bead system containing matrix A (Biomedicals), QIAzol (Qiagen) and Fastprep 120. RNA was extracted with RNeasy (Qiagen). Primers were designed and tested on hepatic tissue lysate, which has been reported to have a high expression of target receptors. Specificity to gene expression was achieved with primers that specifically annealed to cDNA deriving from spliced mRNA expression of Actb, P2X4R, P2X7R and P2Y2R gene. Expression of P2X4 was seen on day 1 in RNA extracts from fractured and non- fractured tibias. Due to technical challenges along the way and the limitation in time, analysis of gene expression in the fracture samples are still pending.

EducationsMolecular Biology, (Bachelor/Graduate Programme) Graduate
LanguageEnglish
Publication date16 Oct 2018
Number of pages73
SupervisorsHenning F. Bjerregaard