An experimental study on the application of radionuclide imaging in repair of the bone defect

Th e aim of our study was to validate the eff ect of radionuclide imaging in early monitoring of the bone’s reconstruction, the animal model of bone defect was made on the rabbits repaired with HA artifi cial bone. Th e ability of bone defect repair was evaluated by using radionuclide bone imaging at , ,  and  weeks postoperatively. Th e results indicate that the experimental group stimulated more bone formation than that of the control group. Th e diff erences of the bone reconstruction ability were statistically signifi cant (p<.). Th e nano-HA artifi cial has good bone conduction, and it can be used for the treatment of bone defects. Radionuclide imaging may be an eff ective and fi rst choice method for the early monitoring of the bone’s reconstruction. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Hydroxyaptite (HA, Ca  (PO)  (OH)  ) shows poor artificial bone mechanics.Its brittleness and low fatigue strength in physiological environment limit its use for load-bearing repair or substitute [].As biomaterials and nanotechnology develop, researchers gradually switch their attention to nano-HA for bone reconstruction purposes.The radionuclide bone imaging is a major technique for observing bone blood supply and metabolism.Because the distribution of radionuclide agents indirectly refl ects the blood supply, metabolism and regeneration of the bone, it sensitively reveals the diseased site and the bone activity at an early stage.Since the s, the radionuclide bone imaging has been used to assess the metabolism of bone minerals.Th e current research explored the ability of novel nano-HA artifi cial bone in repairing the large segmental bone defect of the radius and dynamically monitored the bone regeneration using the radionuclide bone imaging in order to evaluate the value of the radionuclide bone imaging for monitoring the bone reconstruction as a clinical application reference.

Material
Nano-HA Th e novel nano-HA was developed in a joint eff ort by the Powder Metallurgy Research Center of Central South University and our hospital.The nano-HA powder was prepared using calcium nitrate and ammonium dihydrogen phosphate through the sol-gel synthesis method [].The average size of the power was less than nm.Th e Nano-HA artifi cial bone with evenly distributed pores was formulated using nano-HA powder on a wooden mode.Th e pore diameter was -μm and the porosity was over  [].

Radionuclide scanner
The single-photon emission computed tomography (SP-SPECT) scanner was purchased from Pegasys Inc. (Tokyo, Japan).Th e radionuclide bone imaging procedure was conducted in partnership with the Department of Nuclear Medicine, Shenzhen Second People's Hospital (Shenzhen, China).

Preparation of radionuclide agent
Th e radionuclide agent, mTechnetium-methylenediphosphonate (mTc-MDP), was purchased from Guangdong Xi'ai Nucleus Pharmaceutical Center (Guangzhou, China) Animals  conventional male New Zealand white rabbits from a closed colony, ranging from .-.kg were purchased from Southern Medical University's Center of Experimental Animals.Th e rabbits were randomized into the treatment group (the bone defect was repaired with the nano-HA artificial bone) and the control group (the defect was repaired with the HA artifi cial bone) with  rabbits in each.

Establishment of the animal bone defect
Each animal was given an intravenous injection of  mg/ kg ketamine hydrochloride through the ear vein.The left anterior limb was chosen for bone defect establishment [].The animal was locally denuded, sterilized and covered with pads at the operation site.A median incision was made on the radius to expose the radius trunk.Th e radius was sawed at .cm from the proximal side, and at mm from the fi rst incision [, ].Th e fragment with periosteum between two incisions was removed.After the wound was flushed with normal saline, different artificial bones were implanted in animals from diff erent groups, and the wound was sutured layer by layer.Th e operation limb was not fi xated locally and the wound was not bound up.When animals were awake from anesthesia, they were returned to the cage and fed normally. units of penicillin were injected intramuscularly each day for consecutively d post operation.

Radionuclide bone imaging
The rabbits were monitored through radionuclide bone imaging at , , , and  weeks post operation [, ].Four animals were randomly selected at each time point and injected with ketamine hydrochloride through the ear vein.Th e infusion injector was left at the ear vein following anesthesia.Th e animals were fi xated at the animal station and pushed to the SPECT center.Each animal was given an intravenous injection of . MBq/kg bolus mTc-MDP through the injector, and drug leakage was carefully observed. images were collected at a speed of  image per second as the whole-body arterial blood images, i.g the blood-fl ow phase. images at a speed of  images per minute were collected and the one in the middle was selected for comparison.h later, images at a speed of  image per  minutes were collected as the whole-body static images, i.g the static phase.

Image analysis and treatment
The Pegasys Software Package and the image method were applied to identify regions of interest (ROI) (.x.cm) at the intact side and the fractured side.The average counts from ROI at both sides and the intake ratio (average counts at the fractured side/average counts at the intact side) in two groups were recorded.

Statistical analysis
SPSS. was applied to analyze the average counts and the intake ratio between two groups.

General condition
All rabbits revealed normal eating behaviors and activities post operation.No death or infections occurred postoperatively.At  week post operation, the wound healed by first intention, and the stitches wore off automatically.The animals showed unlimited normal walking activities.

Blood-fl ow phase
Th ere was no signifi cant diff erence in the blood-fl ow phase between the treatment group and the control group at different time points.Following injection of radionuclide agents into the vein, the blood-flow images of systematic and local blood circulation were observed instantly, and the contour of organs was then revealed.There was little accumulation in bones with low radioactivity at both the fractured side and the intact side (Figure  and ).Blood-pool phase Radionuclide agents were mostly accumulated in bloodpool images.Soft tissues and organs over the body were clear with increased radioactivity that was distributed evenly images.The radioactivity in bones was low and the bones were not completely revealed (Figure -).In blood-pool images, there was a statistically significant diff erence between the experiment group and the control group.Th e counts from ROI at the fractured side and the uptake ratio increased continually in the two groups at ,  and  weeks, and started to decrease at  weeks.Statistical analysis showed that there were signifi cant diff erences between  groups at diff erent time points (p<.) and within  groups among diff erent time points (P<.).Th e counts from ROI at the fractured side and the uptake ratio reached the peak at  weeks and decreased thereafter (Table  and ).

Static phase
In the static phase, radionuclide agents were mostly accumulated in bones in the treatment group and the control group.Th e bone joints, kidney, and bladder were clearly revealed with obvious radioactivity increase, indicating that most radionuclide agents are excreted through the urinary system.In the treatment group, the counts from ROI at the frac-tured side and the intake ratio increased continually at ,  and  weeks, but started to decrease at  weeks at the fractured side.Th ere was no apparent change in the counts from ROI at the fractured side and the intake ratio at the intact side as time went by.Th e counts from ROI at the fractured side were higher than the intact side at diff erent time points.In the control group, the counts from ROI at the fractured side and the intake ratio increased before  weeks post operation at the fractured side, but not as obvious as in the experiment group.Th ere was a statistically signifi cant diff erence in the counts from ROI at the fractured side, and the intake ratio at ,  and  weeks within the two groups (p <.).The counts    from ROI at the fractured side and the intake ratio increased continually at the fractured side in the two groups as time went by.There was a statistically significant difference in the counts from ROI at the fractured side and the intake ratio at  and  weeks between two groups, and the counts from ROI at the fractured side and the intake ratio at  weeks decreased, compared to that at  weeks.There was a statistically significant difference between the two groups at diff erent time points (, ,  and  weeks) (p<.)(Table -

Development of the HA artifi cial bone
Natural bones have a porous structure that bears forces within a range and maintains smooth blood fl ow ensuring normal growth and metabolism of bone tissues.As bone repair materials, the artifi cial bone should mimic the natural bone structure for bone regeneration.Mimicking, simulating and replicating the porous bone structure to develop bioactive and degradable porous bone repair materials remain a major research focus in biomaterial development.
HA is highly bioactive and biocompatible that tends to well match the nature bone, and is a desirable supplement for bone defect.However, the HA ceramics has poor mechanical features that limit its use in areas of the bone that sustaining forces and thus is mainly applied in bone defect without force bearing.Researchers around the world have researched extensively to improve brittleness of HA.
As the nanotechnology develops, researchers fi nd that HA in the natural bone is mainly nanoscale needle-like monocrystals that align within the collagen matrix in a systematic manner.One study demonstrates that some materials due to "nanoscale phenomenon" may change in terms of mechanic   features when their particles are as small as the nanometer level [].When the particles are small enough, the torsion modulus, tensile modulus and tensile strength are high, and the fatigue resistance also increases.Nano-HPA are believed to have good biological and mechanical features [].Th rough years of research we develop the nano-HA artificial bone through the sol-gel synthesis approach using calcium nitrate and ammonium dihydrogen phosphate, and our testing data demonstrate the material has good biological features [].

Radionuclide bone imaging in bone defect reconstruction
Th e rationale of radionuclide bone imaging is that, following venous injection or oral administration of radionuclide agents and composites, blood circulation, ion exchange and chemical absorption occur between them and positive/negative ions with diff erent valence on the surface of HA, or they may combine to immature collagens that are revealed in imaging, as the bone contains minerals and collagens including HA [, ].Th e two most important factors infl uencing the accumulation of radionuclide agents include () the integrity of blood supply and the rate of bone turnover []; () the bone metabolism, especially the bone regeneration rate; and () affi nity of osteoid and immature collagen with mTc-MDP as well as alkaline phosphatase stimulation [].In recent years, as short half-life radionuclide agents are successfully developed and nuclear instruments and computer technology advance, the radionuclide imaging technique is enhanced and its application is thus expanded, rendering it as a major diagnostic technique for multiple bone and bone joint diseases especially in the early phase.Radionuclide agents accumulate when osteoblasts are active and new bones develop.Smelt et al. reported that the accuracy of prediction using radionuclide bone imaging reached nearly .It has been shown that the radionuclide bone imaging is advantageous over X-ray in early diagnosis of femoral head necrosis, and reveals bone metabolism and bone formation at an early stage that ensures its use in monitoring early bone implant [].
The current research explored radionuclide bone imaging in the blood-fl ow phase, the blood-pool phase and the static phase for reconstructed radius using the nanometer-HA in the rabbit.Th ere is no statistically signifi cant diff erence in the counts from ROI at the fractured side and the uptake ratio between the two groups at diff erent time points in the blood-fl ow phase.An underlying cause is that the blood-fl ow phase mainly refl ects perfusion in blood circulation and the large blood vessels on the bones.As there was no obvious damage to the local blood vessels, thus no signifi cant diff erence is noted in the counts from ROI at the fractured side and the uptake ratio.Th e blood-pool phase mainly refl ects the blood distribution in soft tissues over the body, while the static phase reveals the local bone metabolism.In the current research, there was mTc-MDP accumulation at the bone defect reconstruction site at  weeks, and increased gradually in both two groups, indicating that there is increased vascularization during bone reconstruction, the implant materials are bioactive, the local bone defect site undergoes obvious bone regeneration, and the bone metabolism increases.Th ere is a statistically signifi cant diff erence in the counts from ROI at the fracture side and the uptake ratio at diff erent time points between two groups, showing that nano-HA and normal HA are signifi cantly diff erent in terms of bone reconstruction: the former is better than the latter in vascularization in the bone defect and bone regeneration that advances to be a promising bone repair material.In the current research, the radionuclide accumulation reached the peak at  weeks, and decreased thereafter, which is consistent with the timeline of bone repair and metabolism.

CONCLUSION
Th e results implies that the radionuclide bone imaging is valuable in revealing bone regeneration activity, and SPECT's monitoring function in bone repair promises a vision that the multiphase radionuclide imaging will be clinically used as a sensitive tool to monitor bone defect reconstruction.

FIGURE 1 .
FIGURE 1. Radionuclide bone imaging photo of experimental group (Blood-fl ow phas)

FIGURE 3 - 5 .
FIGURE 3-5.Radionuclide bone imaging photo of experimental group (4 weeks, 8 weeks, 12 weeks postoperatively, Blood-cistern phas, ROI counting level of operative limb increased progressively from 4 to 8 weeks postoperatively and descended 12 weeks postoperatively.ROI counting level of unaff ected limb did not change).

FIGURE 6 - 8 .
FIGURE 6-8.Radionuclide bone imaging photo of control group (4 weeks, 8 weeks, 12 weeks postoperatively, Blood-cistern phas, ROI counting level of operative limb increased progressively from 4 to 8 weeks postoperatively and descended 12 weeks postoperatively.ROI counting level of unaff ected limb did not change).
), and the counts from ROI at the fractured side and the intake ratio at diff erent time points were different between two groups (Figure -).

FIGURE 9 -
FIGURE 9-11.Radionuclide bone imaging photo of experimental group (4 weeks, 8 weeks, 12 weeks postoperatively, Static phase, ROI counting level of operative limb increased progressively from 4 to 8 weeks postoperatively and descended 12 weeks postoperatively.ROI counting level of unaff ected limb did not change).

FIGURE 12 -
FIGURE 12-14.Radionuclide bone imaging photo of control group (4 weeks, 8 weeks, 12 weeks postoperatively, Static phase, ROI counting level of operative limb increased progressively from 4 to 8 weeks postoperatively and descended 12 weeks postoperatively.ROI counting level of unaff ected limb did not change).

TABLE 1 .
Counts from ROI in the blood-pool phase ( X ±S, n=4)

TABLE 2 .
Intake ratio in the blood-pool phase ( X ±S, n=4)

TABLE 3 .
Counts from ROI at the fractured side in the static phase ( X ±S, n=4)

TABLE 4 .
Intake ratio at the fractured side in the static phase ( X ±S, n=4)