Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation

Abstract

Objectives: Orofacial bone is commonly affected by osteoradionecrosis (ORN) during head and neck cancer radiotherapy possibly due to interactions of several factors including radiation damage to resident bone marrow stromal cells (BMSCs). Irradiation causes DNA damage, triggers p53-dependent signalling resulting in either cell-cycle arrest or apoptosis. In same individuals, disproportionately higher rapid growth of orofacial BMSCs relative to those of axial/appendicular bones suggests their response to radiation is skeletally site-specific. We hypothesised that survival and osteogenic recovery capacity of irradiated human BMSCs is site-dependent based on anatomic skeletal site of origin.

Methods: Early passage BMSCs from maxilla, mandible and iliac crest of four normal volunteers were exposed to 2.5 to 10 Gy gamma radiation to evaluate clonogenic survival, effects on cell cycle, DNA damage, p53-related response and in vivo osteogenic regenerative capacity.

Results: Orofacial bone marrow stromal cells (OF-MSCs) survived higher radiation doses and recovered quicker than iliac crest (IC-MSCs) based on clonogenic survival, proliferation and accumulation in G0G1 phase. Post-irradiation p53 level was relatively unchanged but expression of p21, a downstream effector was moderately increased in OF-MSCs. Re-establishment of in vivo bone regeneration was delayed more in irradiated IC-MSCs relative to OF-MSCs.

Conclusions: Effect of irradiation on human BMSCs was skeletal site-specific with OF-MSCs displaying higher radio-resistance and quicker recovery than IC-MSCs.

Figure 1

Disparate site-dependent post-irradiation survival of BMSCs. A. Clonogenic radiation survival analysis showed site and dose-dependent radiobiological response of BMSCs from three skeletal sites. Maxilla and mandible BMSCs were more radio-resistant than iliac crest BMSCs (P < 0.05, triplicate experiments of n = 4 subjects). B and C. Data was fitted to linear quadratic equation, D₀ was calculated from slope, while α and β were transformed using linear survival expression −ln (Surviving fraction/Dose) = α + β(Dose). [D₀ = 1.052 (iliac crest), −0.758 (maxilla) and −0.926 (mandible); ñ = 0.4345 (iliac crest), 1.321 (maxilla) and 4.673 (mandible)]. D. Maxilla and mandible also recovered quicker than iliac crest BMSCs as early as 6 hours post-irradiation (** = P < 0.01).

Figure 2

Cell cycle distribution, DNA damage and p53-related changes in irradiated BMSCs. A. Cell cycle distribution of non-irradiated and irradiated BMSCs from the three skeletal sites indicate radiation-induced G₀G₁ arrest based on more BMSCs accumulation in G₀G₁ post-irradiation. B. Representative images of the comet tails visualized by SYBR Green I staining 30 minutes post-irradiation indicate more DNA damage in iliac crest relative to maxilla and mandible BMSCs (Analysis of relative changes in tail moment of ≥ 50 cells indicate statistically significant differences between IC-MSCs and OF-MSCs, P < 0.01). C and D. Representative Western blots and p53 levels based on densitometric analysis from irradiated cells (n=4 subjects) showed disparate p53 levels based on immunoreactivity to mouse anti-human p53 antibody. E. The response to radiation based on p21^Waf1/Cip1 mRNA was higher in maxilla and mandible BMSCs (n=4 subjects) post-irradiation suggesting underlying mechanisms may be downstream of p53 signaling.

Figure 3

In vivo bone formation by irradiated BMSCs. A. Hematoxylin/eosin-stained sections in vivo bone showed irradiated BMSCs recovered and formed appreciable bone by 12 weeks. B. Despite irradiation, 12 weeks allowed the three cell types to recover and form bone quantitatively similar to non-irradiated cells. (Histology sections representatives of BMSCs transplants from n = 4 subjects; FT = fibrous tissue; carrier = hydroxyapatite/tricalcium phosphate).