A Quantitative µCT and Histomorphometry Analysis of Preferential Radiation Effect on the Endochondral Bone Ossification
Yongren Wu1, Zilan Lin1, Evan L. Hanna1, Robert Holmes1, Daniel G. Mcdonald1,
Kenneth N. Vanek1, Hai Yao2, William R. Barfield1, Vincent D. Pellegrini, Jr1
1
Medical University of South Carolina (MUSC), Charleston, SC, 2Clemson University, Clemson, SC
Disclosures: Yongren Wu (N), Zilan Lin (N), Evan L. Hanna (N), Robert Holmes (N), Daniel G. Mcdonald (N),
Kenneth N. Vanek (N), Hai Yao (N), William R. Barfield (N), Vincent D. Pellegrini, Jr (N)
INTRODUCTION
Due to the radio-sensitivity of the malignant processes in various cancers, external beam irradiation of bony metastatic lesions has become a widely
accepted therapy to prevent further metastasis and subsequent pathologic fracture, and to facilitate bony healing of malignant lesions that compromise the
structural integrity of the skeleton. However, this irradiation potentially interferes with the normal process of bone formation, which is important in healing
of weakened bone. Such are the competing effects of radiation on bone repair. Radiation acts upon both cancerous cells and healthy bone cells and,
depending upon the balance of these effects, may promote or impair normal bone healing after fixation of pathologic fractures. To date, these seemingly
competing effects of radiation on osteogenesis and fracture repair are poorly understood. Based upon clinical observations, Bonarigo and Rubin suggested
that callus formation, as it occurs in endochondral ossification, might be inhibited by radiation. We hypothesize that radiation will impair fractures fixed with
intramedullary nails, which heal primarily by endochondral ossification, more severely than fractures repaired with rigid plates, which heal largely through
intramembranous ossification. The objective of this study was to compare the differential effects of radiation on the two pathways of bone healing in support
of identifying an optimal method of surgical fracture repair of pathologic fractures that require external beam irradiation for local tumor control.
METHODS
Two cohorts of skeletally mature male Sprague-Dawley (SD) rats (n=30) underwent bilateral femur fracture and repair with rigid, compression plating of
one femur and fixation with a custom designed dynamically locked intramedullary nail on the contralateral side (Figure 1). One cohort of animals served as
controls, while the other cohort received external beam irradiation delivered bilaterally on postoperative day 3 at an 8 Gy dose determined to be biologically
equivalent to that used to treat metastatic adenocarcinoma in humans. Animals were euthanized at 1 week, 2 week, 4 week, 3 month and 6 month postoperatively; osseous healing and radiation effects on two distinct healing pathways were quantitatively evaluated using both micro-CT and
histomorphometry. Specifically, micro CT imaging was acquired with 10μm isotropic voxel size and the morphology and microstructure of ossification at
the fracture sites were quantitatively assessed using a Scanco µCT40 system. Two histological techniques: either Safranin O and Fast Green, or Hematoxylin
and Eosin (HE) were utilized to provide clear qualitative differentiation between cartilage, fibrous tissue, and bone at the maximum longitudinal cross
sections of the femurs. Quantification of cartilage at the fracture site was assessed using a commercial histomorphometry software suite. The volumes and
tissue mineral densities of calcified callus and native femur bone, as well as areas of cartilage tissue around the fractures, were examined to detect
differences among groups based on radiation or fixation method during progression of fracture healing. Two-way analysis of variance (ANOVA) or
Student’s t-test were utilized as appropriate. A p-value < 0.05 indicated statistical significance.
RESULTS
In femurs repaired with intramedullary nails, a layer of calcified callus gradually formed around the fracture sites in femurs repaired by nails. A statistical
difference (p=0.042) in the volume of calcified callus was demonstrated between control (39.76±3.50 mm3) and radiation (33.67±8.69 mm3) groups at week
4 time point, representing an approximately 40% decrease in volume of calcified callus in the radiation group (Figure 2). By contrast, in femurs repaired by
plates, a thinner layer of calcified callus was observed around the fracture sites. No statistical difference (p≥0.05) in the volume of calcified callus was
observed between the control and radiation groups at any time points within 6 month (Figure 2). In femurs fixed with nails, our histomorphometry analysis
showed that more cartilage tissue was formed around the fractures in control group (week 2: 37.32±19.88 mm2; week 4: 39.10±16.28 mm2) than that in
radiation group (week 2: 1.54±1.13 mm2; week 4: 4.60±3.97 mm2) at both week 2 and 4 time points (p<0.0001; Figure 3). By contrast, no statistical radiation
effect (p≥0.05) was found on cartilage formation around the fractures in femurs repaired with plates at any time points within 6 month (Figure 3).
DISCUSSION
Based on our micro-CT and histological data, external beam irradiation has a significant and specific deleterious effect on the external calcified callus and
cartilage formation around femoral fractures fixed with intramedullary nails within first month which has been diminished afterwards, while radiation had
little effect on femurs fixed with rigid plates. Likewise, fractures healing by endochondral ossification were preferentially impaired by radiation exposure
and exhibited reduced formation and mineralization of cartilage callus compared to those healing by intramembranous ossification. We hypothesize that
radiation may inhibit neovascularization and mineralization of cartilage callus during its transition to bone during endochondral ossification.
Immunohistological staining techniques will be utilized to further investigate neovascularization and cell proliferation around fractures. Our current
investigations also focus on further elucidating effects of radiation on the progression of bone healing as measured by acquisition of biomechanical strength
up to 6 months post-op.
SIGNIFICANCE
This work represents the successful establishment of a bilateral weight bearing femur fracture model in the Sprague-Dawley rat and has demonstrated a
differential effect of radiation on the two bone healing pathways. These findings suggest that further study is indicated to explore a scientific basis for
consideration of rigid internal fixation via compression plating as the preferred method of fracture repair to drive bone healing down a pathway of
intramembranous ossification in certain biological scenarios. Such indeed may be the case in select pathologic fractures that require adjunctive radiation and
where adjacent bone stock is satisfactory to hold screw fixation and eventual healing of the fracture is desirable for long term durability when extended
survival of the patient is anticipated.
ORS 2017 Annual Meeting Poster No.2194