Phosphate (Pi) is essential for healthy bone growth as well as normal fracture repair. Studies have shown that when animals are phosphate deficient normal fracture healing is interrupted. Although phosphate deficiency has been shown to impair fracture healing, it is unknown how different genetic factors interact with phosphate deficiency to disrupt healing. Furthermore, it is unknown if upon replenishing phosphate in the diet healing will be re-initiated or if the deficiencies will persist irreversibly to prolong the healing of the bones. To assess how genetic factors interact with phosphate deficiency, fractures were generated in three genetically distinct strains of mice that had previously been shown to have different patterns of endochondral bone formation. Phosphate deficiency was initiated two days prior to fracture and was then maintained for a 15 day period covering the normal duration of endochondral bone development. To assess if replenishing phosphate could rescue genetic expression of deficient healing, normal phosphate was re-introduced into the diet after 15 days of deficiency and bone healing was allowed to continue until 35 days post fracture. Messenger RNA expression for marker genes for cartilage and bone formation was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis over this time course of healing. Structural properties, callus mineralization and cartilage contents were assessed by micro-computed tomography and contrast agent enhanced micro- computed tomography (CECT). Torsional mechanical testing was used to measure bone strength. To assess if replenishing phosphate could rescue mineralization and biomechanical properties of deficient healing, normal phosphate was re-introduced after 15 days of deficiency and bone healing was allowed to continue until 21 days post fracture. The biological assessment of fracture healing showed that all three genetic strains had impaired expression of both cartilage and bone associated genes during the period of phosphate deficiency. Once phosphate was returned to the diet, however, the osteogenesis genes showed a burst of late expression in all three strains. Interestingly, torsional testing of the bones showed that phosphate deficient/replenished groups were all stronger but also more brittle than the bones of control mice. Micro-computed tomography demonstrated that bone mineral density was slightly higher in the phosphate deficient mice but the bone mineral density standard deviation in the calluses were also higher indicating that the mineralization within the healing calluses was unevenly distributed in the phosphate deficient/replenished group. Lastly, contrast agent enhanced computed tomography data showed that the overall callus tissue mineral density was lower in phosphate deficient/replenished calluses due to the greater cartilage in the phosphate deficient/replenished calluses. These results suggest that the increase strength in the phosphate deficient/replenished calluses is due to the burst of expression in osteogenesis genes that led to the rapid mineralization of the fracture gap in order to compensate for fracture instability due to the phosphate restriction. They also show that a gross metabolic alteration supersedes all other aspects of genetic variably in endochondral development. Finally, they show that even though fracture healing may be greatly delayed by phosphate deficiency, replacement of phosphate after deficiency leads to rapid regain in function. Future studies need to be carried out to determine if longer time lengths of phosphate deficiency can be rescued upon reintroduction of phosphate.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/14683 |
| Date | 22 January 2016 |
| Creators | Hogue, Brenna |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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