Influence of growth rate on the immature skeleton

Dianne Murray

Research output: ThesisDoctoral Thesis

Abstract

Bone architecture adapts to withstand the loads placed upon it. In response to increased loads during growth, bones circumferentially expand to increase their diameter through the incorporation of periosteal blood vessels and the formation and infilling of primary osteons. However, the influence of growth rate on bone architecture in the immature skeleton is not fully understood. To investigate how bone architecture is modulated by growth rate, morphometric, biochemical and genetic comparisons were made between tibiae from broiler chickens with either fast or slow growth potentials. Both strains of chickens were kept under identical conditions, and fed ad-libitum standard broiler feed. Tibiae were removed and tested by three-point bending to determine stiffness and breaking strength and cross sections of the tibia were examined histomorphologically. Bone stiffness and breaking strength were higher in the rapidly growing birds, but after adjustment for body weight the bones were inherently weaker. Cortical porosity periosteally, but not endosteally, was increased. Sections reacted for ALP and TRAP activity, and others stained for cement (reversal) lines indicated the absence of primary osteon remodelling in the periosteal region. This suggests that the increased periosteal porosity is due to slower infilling of the primary osteons in the rapidly growing birds. To directly quantify the rate of osteon infilling, tibiae were removed from 21-day-old chicks, which had been double labelled with calcein (80 and 8 h before death). The mineral apposition rate was higher in the slow growing chickens, and confirmined the previous histomorphometry results. Osteocyte density within the circumferential lamellae of the cortical bone was higher in the rapidly growing birds but unchanged within the newly laid down bone of the primary osteons. Immunohistochemical staining of cortical bone sections from chickens injected with bromodeoxyuridine located proliferating pre-osteoblast cells to the osteogenic layer of the periosteum. A lower labelling index in the rapidly growing birds was seen across four circumferential areas of the periosteum (anterior = fast growing area, posterior = slow growing area, medial and lateral = intermediate growing areas), even though the osteogenic layer of the periosteum was thicker in the fast strain. Blood vessel numbers within the periosteum was similar between strains but differed between regions habitually loaded in tension (anterior) or in compression (posterior). Osteoblasts were grown and expanded in culture from explants of tibia cortical bone (periosteum removed) of 21-day-old birds of both strains (n=4/strain). Osteoblast proliferation was determined by tritiated-thymidine uptake and differentiation by alkaline phosphatase (ALP) activity. At pre-confluency, cell proliferation was higher in the slow growing birds, but this pattern was reversed at confluency and post confluency which was a likely consequence of impairment of proliferation by contact inhibition in the slow growing strain. ALP activity was only detected at post-confluency and was higher in the fast growing strain. Osteoblastic gene expression was determined by RT-PCR and quantified by densitometry. A higher level of osteopontin, and bone sialoprotein expression (BSP) was observed in the slow growing birds. Whereas Runx2 and the serotonin receptor, considered to have a role in mechanoregulation, were more highly expressed in the fast chickens. In conclusion, fast growth resulted in the expected circumferential expansion to increase bone bending strength. Fast growth was accompanied by increased porosity resulting from the rapid formation of primary osteons and the incapacity of osteoblasts to completely infill the resultant canal. However, periosteal interstitial bone of the fast growing birds had a higher osteocyte density suggesting that the lack of infilling was not due to a decrease in osteoblast number. No evidence was obtained to suggest that osteonal remodelling or periosteal blood vessel number were a determinant for primary osteon size. However, the lower labelling index at the periosteum and increased osteocyte density within the circumferential lamellae of the fast strain suggests an increase in transit time through the osteoblast lineage at the periosteal surface. In vitro, osteoblast proliferation was faster in the slow growing birds whereas differentiation was slower. This is in accord with the previous hypothesis that the fast growing birds are characterised by an increase in transit time through the osteoblast lineage, which may be driven by the high levels of Runx2 expression. Osteopontin and BSP are associated with mechanical loading but the significance of the lower expression levels in the fast growing birds requires further study. However, the up regulation of serotonin expression may reflect the greater loads experienced in the fast growing birds in vivo.

Original languageEnglish
QualificationPh.D.
Awarding Institution
  • University of Edinburgh
Supervisors/Advisors
  • Simpson, Hamish, Supervisor
  • Farquharson, Colin, Supervisor
Award date15 Jul 2005
Publication statusPublished - 2005

Keywords

  • bone
  • skeleton
  • avian
  • chick

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